Multiphase nucleic acid amplification

ABSTRACT

Improved methods for use in nucleic acid amplification, including multiplex amplification, where the amplification is carried out in two or more distinct phases are disclosed. The first phase amplification reaction preferably lacks one or more components required for exponential amplification. The lacking component is subsequently provided in a second, third or further phase(s) of amplification, resulting in a rapid exponential amplification reaction. The multiphase protocol results in faster and more sensitive detection and lower variability at low analyte concentrations. Compositions for carrying out the claimed methods are also disclosed.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/695,106, filed Aug. 30, 2012; and U.S. Provisional Application No.61/846,538, filed Jul. 15, 2013. The entire disclosures of these earlierapplications are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention generally relates to the field of molecular biology. Morespecifically, the invention relates to multiphase in vitro amplificationof nucleic acids, which is useful for increasing the efficiency andprecision of amplification in both uniplex and multiplex reactions,allowing a more sensitive detection of nucleic acid targets withimproved quantitation characteristics as well as less interferencebetween analytes in multiplex reactions.

BACKGROUND OF THE INVENTION

Nucleic acid amplification provides a means for generating multiplecopies of a nucleic acid sequence that is relatively rare or unknown,for identifying the source of nucleic acids, or for making sufficientnucleic acid to provide a readily detectable amount. Amplification isuseful in many applications, for example, in sequencing, diagnostics,drug development, forensic investigations, environmental analysis, andfood testing. Many methods for amplifying nucleic acid sequences invitro are known, including polymerase chain reaction (PCR), ligase chainreaction (LCR), replicase-mediated amplification, strand-displacementamplification (SDA), “rolling circle” types of amplification, andvarious transcription associated amplification methods. These knownmethods use different techniques to make amplified sequences, whichusually are detected by using a variety of methods.

PCR amplification uses a DNA polymerase, oligonucleotide primers, andthermal cycling to synthesize multiple copies of both strands of adouble-stranded DNA (dsDNA) or dsDNA made from a cDNA (U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159). LCR amplification uses an excessof two complementary pairs of single-stranded probes that hybridize tocontiguous target sequences and are ligated to form fused probescomplementary to the original target, which allows the fused probes toserve as a template for further fusions in multiple cycles ofhybridization, ligation, and denaturation (U.S. Pat. No. 5,516,663 andEP 0320308 B1). Replicase-mediated amplification uses a self-replicatingRNA sequence attached to the analyte sequence and a replicase, such asQβ-replicase, to synthesize copies of the self-replicating sequencespecific for the chosen replicase, such as a Qβ viral sequence (U.S.Pat. No. 4,786,600). The amplified sequence is detected as a substituteor reporter molecule for the analyte sequence. SDA uses a primer thatcontains a recognition site for a restriction endonuclease which allowsthe endonuclease to nick one strand of a hemi-modified dsDNA thatincludes the target sequence, followed by a series of primer extensionand strand displacement steps (U.S. Pat. Nos. 5,422,252 and 5,547,861).Rolling circle types of amplification rely on a circular or concatenatednucleic acid structure that serves as a template used to enzymaticallyreplicate multiple single-stranded copies from the template (e.g., U.S.Pat. Nos. 5,714,320 and 5,834,252). Transcription-associatedamplification refers to methods that amplify a sequence by producingmultiple transcripts from a nucleic acid template. Such methodsgenerally use one or more oligonucleotides, of which one provides apromoter sequence, and enzymes with RNA polymerase and DNA polymeraseactivities to make a functional promoter sequence near the targetsequence and then transcribe the target sequence from the promoter(e.g., U.S. Pat. Nos. 4,868,105, 5,124,246, 5,130,238, 5,399,491,5,437,990, 5,554,516 and 7,374,885; and PCT Pub. No. WO 1988/010315).

Nucleic acid amplification methods may amplify a specific targetsequence (e.g., a gene sequence), a group of related target sequences,or a surrogate sequence, which may be referred to as a tag sequence.This tag sequence can be used for a variety of purposes, such asdetection, further amplification, as a binding tag for immobilization,as an adaptor for use in various functions in sequencing reactions,including next generation sequencing, etc. The tag sequence isfunctional for its intended purpose only if the analyte target sequenceis present at some point during the reaction. Modified nucleic acidamplification methods may amplify more than one potential targetsequence by using “universal” primer(s) or universal priming. One formof PCR amplification uses universal primers that bind to conservedsequences to amplify related sequences in a PCR reaction (Okamoto etal., 1992, J. Gen. Virol. 73:673-679, Persing et al., 1992, J. Clin.Microbiol. 30:2097-2103). Methods that use universal primers often arepaired with use of a species-specific, gene-specific or type-specificprimer or primers to generate an amplified sequence that is unique to aspecies, genetic variant, or viral type, which may be identified bysequencing or detecting some other characteristic of the amplifiednucleic acid. Anchored PCR is another modified PCR method that uses auniversal primer or an “adapter” primer to amplify a sequence that isonly partially known. Anchored PCR introduces an “adaptor” or“universal” sequence into a cDNA and then uses a primer that binds tothe introduced sequence in subsequent amplification steps. Generally,anchored-PCR uses a primer directed to a known sequence to make a cDNA,adds a known sequence (e.g., poly-G) to the cDNA or uses a commonsequence in the cDNA (e.g., poly-T), and performs PCR by using auniversal primer that binds to the added or common sequence in the cDNAand a downstream target-specific primer (Loh et al., 1989, Science243:217-220; Lin et al., 1990, Mol. Cell. Biol. 10:1818-1821). NestedPCR may use primer(s) that contain a universal sequence unrelated to theanalyte target sequence to amplify nucleic acid from unknown targetsequences in a reaction (Sullivan et al., 1991, Electrophoresis12:17-21; Sugimoto et al., 1991, Agric. Biol. Chem. 55:2687-2692).

Chamberlain et al. (Nucleic Acid Res., 1988, 16:11141-11156) firstdemonstrated multiplex PCR analysis for the human dystrophin gene.Multiplex reactions are accomplished by careful selection andoptimization of specific primers. Developing robust, sensitive andspecific multiplex reactions have demanded a number of specific designconsiderations and empiric optimizations. This results in longdevelopment times and compromises reaction conditions that reduce assaysensitivity. In turn, development of new multiplex diagnostic testsbecomes very costly. A number of specific problems have been identifiedthat limit multiplex reactions. Incorporating primer sets for more thanone target requires careful matching of the reaction efficiencies. Ifone primer amplifies its target with even slightly better efficiency,amplification becomes biased toward the more efficiently amplifiedtarget resulting in inefficient amplification, varied sensitivity andpossible total failure of other target genes in the multiplex reaction.This is called “preferential amplification.” Preferential amplificationcan sometimes be corrected by carefully matching all primer sequences tosimilar lengths and GC content and optimizing the primer concentrations,for example by increasing the primer concentration of the less efficienttargets. Incorporation of inosine into primers in an attempt to adjustthe primer amplification efficiencies also has been used (U.S. Pat. No.5,738,995). Another approach is to design chimeric primers, where eachprimer contains a 3′ region complementary to sequence-specific targetrecognition and a 5′ region made up of a universal sequence. Using theuniversal sequence primer permits the amplification efficiencies of thedifferent targets to be normalized (Shuber et al., Genome Res., 1995,5:488-493; and U.S. Pat. No. 5,882,856). Chimeric primers have also beenutilized to multiplex isothermal strand displacement amplification (U.S.Pat. Nos. 5,422,252, 5,624,825 and 5,736,365).

Since multiple primer sets are present in multiplex amplificationreactions, multiplexing is frequently complicated by artifacts resultingfrom cross-reactivity of the primers. All possible combinations must beanalyzed so that as the number of targets increases this becomesextremely complex and severely limits primer selection. Even carefullydesigned primer combinations often produce spurious products that resultin either false negative or false positive results. The reactionkinetics and efficiency is altered when more than one reaction isoccurring simultaneously. Each multiplexed reaction for each differentspecimen type must be optimized for MgCl₂ concentration and ratio to thedeoxynucleotide concentration, KCl concentration, amplification enzymeconcentration, and amplification reaction times and temperatures. Thereis a competition for the reagents in multiplex reactions so that all ofthe reactions plateau earlier. As a consequence, multiplexed reactionsin general are less sensitive and often prone to more variability thanthe corresponding uniplex reaction.

Another consideration to simultaneous amplification reactions is thatthere must be a method for the discrimination and detection of each ofthe targets. The number of multiplexed targets is then further limitedby the number of dye or other label moieties distinguishable within thereaction. As the number of different fluorescent moieties to be detectedincreases, so does the complexity of the optical system and dataanalysis programs necessary for result interpretation. One approach isto hybridize the amplified multiplex products to a solid phase thendetect each target. This can utilize a planar hybridization platformwith a defined pattern of capture probes (U.S. Pat. No. 5,955,268), orcapture onto a bead set that can be sorted by flow cytometry (U.S. Pat.No. 5,981,180).

Due to the multitude of the technical issues discussed, currenttechnology for multiplex gene detection is costly and severely limitedin the number and combinations of genes that can be analyzed. Generally,these reactions multiplex only two or three targets with a maximum ofaround ten targets. Isothermal amplification reactions are more complexthan PCR and even more difficult to multiplex (Van Deursen et al.,Nucleic Acid Res., 1999, 27:e15). U.S. Pat. No. 6,605,451 discloses atwo-step PCR multiplex reaction where a small amount of each primer pairis added into a first PCR reaction mix, and a first exponentialamplification is performed to increase the amount of target nucleicacids in the reaction. The first reaction is stopped mid log phase andis then separated into second reactions each containing primer pairs forone of the target nucleic acids. A full exponential amplification isthen performed. Though a limited amount of each of the multiplex primerpairs is present in the first reaction, the above discussed problemscommon to multiplexing are still present. Further, these various primerpair species can all transfer into the secondary amplificationreactions, causing common multiplex problems there as well.

Accordingly, there is still a need for a method which permitsmultiplexing of large numbers of targets without extensive design andoptimization constraints, and which avoids problems common tomultiplexing in the presence of a plurality of different amplificationoligonucleotide pairs. In addition, there is an ongoing need to improvesensitivity and precision at the limit of detection and/orquantification for both multiplex and uniplex amplification reactions.The present invention addresses these and other needs.

SUMMARY OF THE INVENTION

As practiced in the art, all components required for nucleic acidamplification are present in the reaction mixture when amplification isstarted (herein referred to as the “single-phase method”). Thissingle-phase method creates a problem in that undesired side reactionsare usually initiated along with the desired amplification reaction.These side reactions often compete with and thus degrade the overallperformance of the desired amplification reaction. Moreover, inmultiplex amplification reactions, amplification of analytes that arepresent at higher amounts in the reaction mixture or analytes whoseoverall amplification efficiency is higher than that of other analytesunduly compete with and thus degrade the amplification of the otheranalytes in the mixture.

The improved method disclosed herein addresses these problems byconducting the amplification reaction in multiple phases. Using thismethod, the desired reaction or reactions are initiated and allowed toproceed to a certain level, whereas initiation or progression of othercompeting reactions is not supported by the reaction conditions. In thisway, the desired reactions in essence get a head start on competingreactions, resulting in improved overall performance of the desiredreactions. Furthermore, multiplex amplification reactions can berendered less competitive with one another by conducting the overallamplification process in phases. Thus, similar to the situationdescribed above, the lower efficiency reactions and/or thosecorresponding to lower initial levels of target analyte are allowed toproceed without the other reactions progressing unchecked and severelyout-competing them. It is contemplated that the general principle ofmultiphase amplification is broadly applicable to a variety ofamplification techniques and can be embodied in a wide variety ofdifferent modes, as described in more detail herein.

Accordingly, in the first aspect, the present invention provides amethod for amplifying a target nucleic acid sequence in a sampleincluding at least two steps. Initially, the target nucleic acidsequence is subjected to a first phase amplification reaction underconditions that do not support exponential amplification of the targetnucleic acid sequence. The first phase amplification reaction generatesa first amplification product, which is subsequently subjected to asecond phase amplification reaction under conditions allowingexponential amplification of the first amplification product, therebygenerating a second amplification product.

As noted above, in many cases, it is desirable to detect and quantifymultiple target nucleic acid sequences in the same sample. Accordingly,in a second aspect, the present invention provides a method foramplifying a plurality of different target nucleic acid sequences in asample including at least two steps. Initially, the target nucleic acidsequences are subjected to a first phase amplification reaction underconditions that do not support, or that prevent, exponentialamplification of any of the target nucleic acid sequences. The firstphase amplification reaction generates a plurality of firstamplification products, which are subsequently subjected to a secondphase amplification reaction under conditions allowing exponentialamplification of the first amplification products, thereby generating aplurality of second amplification products.

In a modified version of the second aspect, the invention provides amethod for amplifying a plurality of different target nucleic acidsequences in a sample, where some, but not all, of the target nucleicacid sequences are subjected to linear amplification, and/or some, butnot all, of the target nucleic acid sequences are subjected toexponential amplification in at least one phase of the reaction.Accordingly, at least four possible variants of the first phaseamplification are contemplated: (1) some of the target sequences aresubjected to linear amplification, and the rest are left unamplified;(2) some of the target sequences are subjected to exponentialamplification, and the rest are left unamplified; (3) some of the targetsequences are subjected to linear amplification, some are subjected toexponential amplification and the rest are left unamplified; and (4)some of the target sequences are subjected to linear amplification andthe rest are subjected to exponential amplification. Thus, in thisaspect of the invention, the first phase amplification may result inamplification of all of the target nucleic acid sequences (option 4) oronly a subset thereof (options 1-3). The subset of the target nucleicacid sequences may represent targets known to be present in relativelylow quantities and/or targets that are difficult to amplify compared toother targets. The first phase amplification reaction generates one ormore first amplification product(s). The first amplification product(s)and any unamplified target nucleic acid sequence(s) in the sample arethen subjected to a second phase amplification reaction under conditionsallowing exponential amplification thereof, generating a plurality ofsecond amplification products. Alternatively, there can be more than twophases where conditions 1-4 above may apply for all phases except thefinal phase and where for the last phase any unamplified or linearlyamplified target nucleic acid sequence(s) in the sample are subjected toan amplification reaction under conditions allowing exponentialamplification thereof.

In a third aspect, the invention provides a composition for amplifying atarget nucleic acid sequence in a sample. The composition includes thefollowing components: (a) an amplification oligonucleotide thathybridizes to a first portion of the target nucleic acid sequence; (b)an optional target capture oligonucleotide that hybridizes to a secondportion of the target nucleic acid sequence; and (c) an amplificationenzyme. One of the key features of the present composition is that itlacks at least one component required for exponential amplification ofthe target nucleic acid sequence. As explained in detail elsewhere inthis application, one of the advantages of the present composition isthat it helps to reduce non-specific amplification, thereby focusing theamplification resources on the target sequence.

In a fourth aspect, the invention provides an alternative compositionfor amplifying a plurality of different target nucleic acid sequences ina sample. The composition includes the following components: (a) aplurality of different amplification oligonucleotide complexes thathybridize to a plurality of different target nucleic acid sequences,where each amplification oligonucleotide complex includes a firstamplification oligonucleotide having a first target specific sequencethat is joined to a second amplification oligonucleotide having a secondtarget specific sequence; (b) a target capture oligonucleotide thathybridizes to a second portion of the target nucleic acid, and (c) anamplification enzyme. Once again, the composition lacks at least onecomponent required for exponential amplification of the target nucleicacid sequences.

In a fifth aspect, the invention provides a method of quantifying atarget nucleic acid sequence in a sample. In accordance with the method,first there is the step of (a) contacting the sample with a firstamplification oligonucleotide, specific for a first portion of thetarget nucleic acid sequence, under conditions allowing hybridization ofthe first amplification oligonucleotide to the first portion of thetarget nucleic acid sequence, thereby generating a pre-amplificationhybrid that includes the first amplification oligonucleotide and thetarget nucleic acid sequence. Next, there is the step of (b) isolatingthe pre-amplification hybrid by target capture onto a solid supportfollowed by washing to remove any of the first amplificationoligonucleotide that did not hybridize to the first portion of thetarget nucleic acid sequence in step (a). This is followed by the stepof (c) amplifying, in a first phase amplification reaction mixture, atleast a portion of the target nucleic acid sequence of thepre-amplification hybrid isolated in step (b) in a first phase,substantially isothermal, transcription-associated amplificationreaction under conditions that support linear amplification thereof, butdo not support exponential amplification thereof, thereby resulting in areaction mixture including a first amplification product. Generallyspeaking, the first phase amplification reaction mixture includes asecond amplification oligonucleotide, the second amplificationoligonucleotide being complementary to a portion of an extension productof the first amplification oligonucleotide. As well, the firstamplification product is not a template for nucleic acid synthesisduring the first phase, substantially isothermal,transcription-associated amplification reaction. Next, there is the stepof (d) combining the reaction mixture including the first amplificationproduct with at least one component that participates in exponentialamplification of the first amplification product, but that is lackingfrom the reaction mixture that includes the first amplification product,to produce a second phase amplification reaction mixture. Generally, thesecond phase amplification reaction mixture additionally includes asequence-specific hybridization probe. Next, there is the step (e) ofperforming, in a second phase, substantially isothermal,transcription-associated amplification reaction in the second phaseamplification reaction mixture, an exponential amplification of thefirst amplification product, thereby synthesizing a second amplificationproduct. This is followed by the steps of (f) detecting, with thesequence-specific hybridization probe at regular time intervals,synthesis of the second amplification product in the second phaseamplification reaction mixture; and then (g) quantifying the targetnucleic acid sequence in the sample using results from the detectingstep (f). In one generally preferred method, the first amplificationoligonucleotide includes a 3′ target specific sequence and a 5′ promotersequence for an RNA polymerase. In a preferred case, the RNA polymeraseis T7 RNA polymerase. In another generally preferred method, the secondamplification oligonucleotide is enzymatically extended in the firstphase isothermal transcription-associated amplification reaction. Inanother generally preferred method, the solid support includes animmobilized capture probe. In another generally preferred method, step(a) further includes contacting the sample with a target captureoligonucleotide that hybridizes to the target nucleic acid sequence; andthe pre-amplification hybrid includes the target nucleic acid sequencehybridized to each of the target capture oligonucleotide and the firstamplification oligonucleotide. In another generally preferred method,the solid support includes magnetically attractable particles. Inanother generally preferred method, each of the first and second phaseisothermal transcription-associated amplification reactions include anRNA polymerase and a reverse transcriptase, and the reversetranscriptase includes an endogenous RNaseH activity. In anothergenerally preferred method, the at least one component includes thefirst amplification oligonucleotide. In another generally preferredmethod, the first amplification product of step (c) is a cDNA moleculewith the same polarity as the target nucleic acid sequence in thesample, and the second amplification product of step (e) is an RNAmolecule. In another generally preferred method, the sequence-specifichybridization probe in step (d) is a conformation-sensitive probe thatproduces a detectable signal when hybridized to the second amplificationproduct. In another generally preferred method, the sequence-specifichybridization probe in step (d) is a fluorescently labeledsequence-specific hybridization probe. In another generally preferredmethod, step (g) includes quantifying the target nucleic acid sequencein the sample using a linear calibration curve and results from step(f). In another generally preferred method, step (c) includes amplifyingby 10-fold to 10,000-fold, in the first phase amplification reactionmixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of dual-phase forwardTranscription-Mediated Amplification (TMA). In this embodiment, anamplification primer containing a T7 promoter (“T7 primer”) hybridizesto a target nucleic acid sequence during target capture, followed byremoval of excess T7 primer. The amplification process is divided intoat least two distinct phases. During the first phase, a non-T7 primer isintroduced along with all of the requisite amplification and enzymereagents (AR and ER, respectively), with the exception of additional T7primer (RT: reverse transcriptase; T7: T7 RNA polymerase). In thepresence of reverse transcriptase, the T7 primer hybridized to thetarget is extended, creating a cDNA copy, and the target RNA template isdegraded by RNase H activity of RT. The non-T7 primer subsequentlyhybridizes to the cDNA and is then extended, filling in the promoterregion of the T7 primer and creating an active, double-strandedtemplate. The T7 polymerase then produces multiple RNA transcripts fromthe template. The non-T7 primer subsequently hybridizes to the RNAtranscripts and is extended, producing promoterless cDNA copies of thetarget RNA template. The RNA strands are then degraded by RNase activityof RT. Because no T7 primer is available in the phase 1 amplificationmixture, the reaction cannot proceed any further. The second phase isthen started with the addition of T7 primer, thus initiating exponentialamplification of the cDNA pool produced in phase 1.

FIGS. 2A-2B show a comparison between the standard single-phase forwardTMA (FIG. 2A) and a modified single-phase forward TMA that was used as acontrol in some of the working examples described herein (FIG. 2B). Thestandard single-phase forward TMA protocol is well-known in the art andis described in detail elsewhere in the present application. In themodified single-phase TMA protocol, a T7 primer hybridizes to a targetnucleic acid sequence during target capture, thereby eliminating theusual T7 primer annealing step at 60° C. following the target capture.Subsequently, a non-T7 primer is added along with additional T7 primerand all of the requisite amplification and enzyme reagents, thusallowing exponential amplification to proceed. As one can see from FIGS.2A and 2B, both of the single-phase TMA protocols appear to havecomparable sensitivities at the low end, reliably detecting 100 or morecopies of a human immunodeficiency virus 1 (HIV-1) target template butperforming poorly at 10 copies of the target template.

FIGS. 3A-3C demonstrate a comparison between the modified single-phaseforward TMA and dual-phase forward TMA. In FIG. 3A, modifiedsingle-phase TMA as described above was used to amplify an HIV-1 targettemplate. In FIG. 3B, the same HIV-1 template was amplified using thedual-phase TMA technique described in FIG. 1, i.e. the T7 primer wasinitially withheld from the amplification mixture and was provided inthe second phase to start exponential amplification. FIG. 3C depictscalibration curve linear fits, showing significant shifts in sensitivitybetween the single-phase and dual-phase amplification reactions.

FIGS. 4A-4D illustrate optimization of the concentration of T7 primeradded in the second phase of amplification. In FIG. 4A, the modifiedsingle-phase forward TMA was used to amplify an HIV-1 target template.In FIGS. 4B-4D, the T7 primer was withheld from the amplificationmixture in phase 1, and different concentrations of T7 primer wereprovided in the second phase to initiate exponential amplification.Comparable significant shifts in sensitivity and robustness between thesingle-phase and dual-phase amplification reactions were observed at 1pmol/r×n, 5 pmol/r×n and 10 pmol/r×n T7 primer.

FIGS. 5A-5D show optimization of the concentration of non-T7 primeradded in the first phase of amplification. In FIG. 5A, the modifiedsingle-phase forward TMA was used to amplify an HIV-1 target template.In FIGS. 5B-5D, T7 primer was withheld from the amplification mixture inphase 1 and was provided in the second phase to initiate exponentialamplification. Comparable significant shifts in sensitivity androbustness between the single-phase and dual-phase amplificationreactions were observed at 10 pmol/r×n and 15 pmol/r×n of non-T7 primer.The shift was somewhat less pronounced in the presence of 2 pmol/r×nnon-T7 primer.

FIGS. 6A-6C demonstrate the effect of the enzyme reagent (RT and T7 RNApolymerase) in the second phase amplification on the overall assayperformance. In FIG. 6A, the modified single-phase forward TMA was usedto amplify an HIV-1 target template. As before, in FIGS. 6B-6C, the T7primer was withheld from the amplification mixture in phase 1 and wasprovided in the second phase to initiate exponential amplification. InFIG. 6B, equal amounts of the enzyme reagent were added in the first andsecond phases of the amplification reaction. In FIG. 6C, the same totalamount of the enzyme reagent was added in the first phase amplification,whereas no enzyme reagent was added in the second phase. Comparablesignificant shifts in sensitivity and robustness between thesingle-phase and dual-phase amplification reactions were observed inboth experiments.

FIGS. 7A-7C show a comparison between the standard single-phase (FIG.7A) and dual-phase (FIG. 7B) forward TMA using a human papillomavirussubtype 16 (HPV16) target template. As discussed above in reference toHIV-1 target template, in the dual-phase format, the T7 primer waswithheld from the amplification mixture in phase 1 and was provided inthe second phase to initiate exponential amplification. FIG. 7C depictscalibration curve linear fits, demonstrating a significant shift insensitivity between the single-phase and dual-phase amplificationreactions.

FIGS. 8A-8C show a comparison between the standard single-phase (FIG.8A) and dual-phase (FIG. 8B) forward TMA using a prostate cancer antigen3 (PCA3) target template. As discussed above in reference to HIV-1 andHPV16 target templates, in the dual-phase format, the T7 primer waswithheld from the amplification mixture in phase 1 and was provided inthe second phase to intiate exponential amplification. FIG. 8C depictscalibration curve linear fits, similarly showing a significant shift insensitivity between the single-phase and dual-phase amplificationreactions.

FIGS. 9A-9F show a comparison between the standard single-phase (FIGS.9A and 9D) and dual-phase (FIGS. 9B and 9E) forward TMA used for duplexamplification of PCA3 and T2-ERG, a prostate cancer marker formed bygene fusion of the androgen-regulated transmembrane serine protease(TMPRSS2) with the ETS transcription factor (ERG). FIGS. 9A and 9B showdetection of PCA3 in the presence of T2-ERG, whereas FIGS. 9D and 9Eshow detection of T2-ERG in the presence of PCA3. FIGS. 9C and 9F depictcalibration curve linear fits, showing significant shifts insensitivities between the single-phase and dual-phase amplificationreactions for both targets in the duplex amplification context.

FIGS. 10A-10I show a comparison between the standard single-phase (FIGS.10A, 10D and 10G) and dual-phase (FIGS. 10B, 10E and 10H) forward TMAused for triplex amplification of PCA3, T2-ERG and prostate specificantigen (PSA). FIGS. 10A and 10B show detection of PCA3 in the presenceof T2-ERG and PSA; FIGS. 10D and 10E show detection of T2-ERG in thepresence of PCA3 and PSA; and FIGS. 10G and 10H show detection of PSA inthe presence of PCA3 and T2-ERG. FIGS. 10C, 10F and 10I depictcalibration curve linear fits, showing significant shifts insensitivities between the single-phase and dual-phase amplificationreactions for all three targets in the triplex amplification context.

FIGS. 11A-11C demonstrate a comparison between a modified single-phasereverse TMA and dual-phase reverse TMA. In FIG. 11A, a modifiedsingle-phase reverse TMA was used to amplify a T2-ERG target template.In the modified single-phase reverse TMA, a non-T7 primer hybridizes toa target nucleic acid sequence during target capture, therebyeliminating the usual non-T7 primer annealing step at 60° C. followingthe target capture. Subsequently, a T7 primer is added along withadditional non-T7 primer and all of the requisite amplification,detection and enzyme reagents, thus allowing exponential amplificationto proceed. In FIG. 11B, the same T2-ERG template was amplified using adual-phase reverse TMA protocol, where the non-T7 primer was withheldfrom the amplification mixture in phase 1 and was provided in the secondphase to initiate exponential amplification. FIG. 11C depictscalibration curve linear fits, showing significant shifts in sensitivitybetween the single-phase and dual-phase amplification reactions.

FIGS. 12A-12C show a comparison between the modified single-phase (FIG.12A), the dual-phase format described in 11 (FIG. 12B), and a differentdual-phase format (FIG. 12C) reverse TMA used for quadruplexamplification of T2-ERG, PCA3, PSA and an internal control (CAP). FIGS.12A, 12B and 12C show detection of T2-ERG in the presence of PCA3, PSAand CAP. In FIG. 12B, all four targets were subjected to the samedual-phase reverse TMA as the one described above in connection withFIG. 11B. In FIG. 12C, PCA3, PSA and CAP (or CAP alone) were subjectedto linear amplification and T2-ERG was subjected to exponentialamplification in the first phase of the reaction, and PCA3, PSA and CAPwere subjected to exponential amplification and T2-ERG continuedamplifying exponentially in the second phase (all of the amplificationreactions proceeded in the same vessel).

FIGS. 13A-13C show a comparison between the modified single-phase (FIG.13A), dual-phase (FIG. 13B), and triple-phase (FIG. 13C) reverse TMAused for quadruplex amplification of T2-ERG, PCA3, PSA, and an internalcontrol (CAP). FIGS. 13A, 13B and 13C show detection of T2-ERG in thepresence of PCA3, PSA and CAP. In FIG. 13B, all four targets weresubjected to the same dual-phase reverse TMA as the one described abovein connection with FIG. 11B. In FIG. 13C, T2-ERG was subjected to linearamplification and the other 3 analytes were not amplified in phase 1,T2-ERG was subjected to exponential amplification and the 3 otheranalytes were not amplified in phase 2, and PCA3, PSA and CAP (or CAPalone) were subjected to exponential amplification and T2-ERG continuedamplifying exponentially in phase 3 (all of the amplification reactionsproceeded in the same vessel).

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. Definitions

Unless otherwise described, scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in the artof molecular biology based on technical literature, e.g., Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), or other well-known technicalpublications related to molecular biology. Unless otherwise described,techniques employed or contemplated herein are standard methods wellknown in the art of molecular biology. To aid in understanding aspectsof the disclosed methods and compositions, some terms are described inmore detail or illustrated by embodiments described herein.

All patents, applications, published applications and other publicationsreferred to herein are incorporated by reference in their entireties. Ifa definition set forth in this section is contrary to or otherwiseinconsistent with a definition set forth in the patents, applications,published applications and other publications that are hereinincorporated by reference, the definition set forth in this sectionprevails over the definition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

Approximating language, as used herein throughout the specification andclaims, may be applied to modify any quantitative or qualitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” or “approximately” is not to belimited to the precise value specified, and may include values thatdiffer from the specified value.

As used herein, the term “sample” refers to a specimen that may containan analyte of interest, e.g., microbe, virus, nucleic acid such as agene, or components thereof, which includes nucleic acid sequences in orderived from an analyte. Samples may be from any source, such asbiological specimens or environmental sources. Biological specimensinclude any tissue or material derived from a living or dead organismthat may contain an analyte or nucleic acid in or derived from ananalyte. Examples of biological samples include respiratory tissue,exudates (e.g., bronchoalveolar lavage), biopsy, sputum, peripheralblood, plasma, serum, lymph node, gastrointestinal tissue, feces, urine,or other fluids, tissues or materials. Examples of environmental samplesinclude water, ice, soil, slurries, debris, biofilms, airborneparticles, and aerosols. Samples may be processed specimens ormaterials, such as obtained from treating a sample by using filtration,centrifugation, sedimentation, or adherence to a medium, such as matrixor support. Other processing of samples may include treatments tophysically or mechanically disrupt tissue, cellular aggregates, or cellsto release intracellular components that include nucleic acids into asolution which may contain other components, such as enzymes, buffers,salts, detergents and the like.

As used herein, the term “contacting” means bringing two or morecomponents together. Contacting can be achieved by mixing all thecomponents in a fluid or semi-fluid mixture. Contacting can also beachieved when one or more components are brought into physical contactwith one or more other components on a solid surface such as a solidtissue section or a substrate.

As used herein, the term “nucleic acid” refers to a polynucleotidecompound, which includes oligonucleotides, comprising nucleosides ornucleoside analogs that have nitrogenous heterocyclic bases or baseanalogs, covalently linked by standard phosphodiester bonds or otherlinkages. Nucleic acids include RNA, DNA, chimeric DNA-RNA polymers oranalogs thereof. In a nucleic acid, the backbone may be made up of avariety of linkages, including one or more of sugar-phosphodiesterlinkages, peptide-nucleic acid (PNA) linkages (PCT Pub No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties in a nucleic acid may be ribose, deoxyribose, orsimilar compounds with substitutions, e.g., 2′ methoxy and 2′ halide(e.g., 2′-F) substitutions. Nitrogenous bases may be conventional bases(A, G, C, T, U), analogs thereof (e.g., inosine; The Biochemistry of theNucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), derivatives ofpurine or pyrimidine bases (e.g., N4-methyl deoxygaunosine, deaza- oraza-purines, deaza- or aza-pyrimidines, pyrimidines or purines withaltered or replacement substituent groups at any of a variety ofchemical positions, e.g., 2-amino-6-methylaminopurine, O6-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines, orpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine (e.g., U.S. Pat. Nos. 5,378,825, 6,949,367 andPCT Pub. No. WO 93/13121)). Nucleic acids may include “abasic” positionsin which the backbone does not have a nitrogenous base at one or morelocations (U.S. Pat. No. 5,585,481), e.g., one or more abasic positionsmay form a linker region that joins separate oligonucleotide sequencestogether. A nucleic acid may comprise only conventional sugars, bases,and linkages as found in conventional RNA and DNA, or may includeconventional components and substitutions (e.g., conventional baseslinked by a 2′ methoxy backbone, or a polymer containing a mixture ofconventional bases and one or more analogs). The term includes “lockednucleic acids” (LNA), which contain one or more LNA nucleotide monomerswith a bicyclic furanose unit locked in a RNA mimicking sugarconformation, which enhances hybridization affinity for complementarysequences in ssRNA, ssDNA, or dsDNA (Vester et al., 2004, Biochemistry43(42): 13233-41).

As used herein, the interchangeable terms “oligonucleotide” and“oligomer” refer to nucleic acid polymers generally made of less than1,000 nucleotide (nt), including those in a size range having a lowerlimit of about 2 to 5 nt and an upper limit of about 500 to 900 nt.Preferred oligonucleotides are in a size range having a 5 to 15 nt lowerlimit and a 50 to 500 nt upper limit, and particularly preferredembodiments are in a size range having a 10 to 20 nt lower limit and a25 to 150 nt upper limit. Preferred oligonucleotides are madesynthetically by using any well-known in vitro chemical or enzymaticmethod, and may be purified after synthesis by using standard methods,e.g., high-performance liquid chromatography (HPLC). Representativeoligonucleotides discussed herein include priming oligonucleotides(e.g., primers, nonT7 primers, T7 promoter-primers, etc.), promoterproviders (which are promoter primers comprising a blocked 3′-end),detection probe oligonucleotides, target capture oligonucleotides, andblockers, to name a few. Priming oligonucleotides and promoter providersare generally referred to as “amplification oligonucleotides.”

A “priming oligonucleotide” (or more simply, “primer”) is anoligonucleotide, at least the 3′-end of which is complementary to anucleic acid template, and which complexes (by hydrogen bonding orhybridization) with the template to give a primer:template complexsuitable for initiation of synthesis by an RNA- or DNA-dependent DNApolymerase. A priming oligonucleotide is extended by the addition ofcovalently bonded nucleotide bases to its 3′-terminus, which bases arecomplementary to the template. The result is a primer extension product.A priming oligonucleotide of the present invention is typically at least10 nucleotides in length, and may extend up to 15, 20, 25, 30, 35, 40,50 or more nucleotides in length. Suitable and preferred primingoligonucleotides are described herein. Virtually all DNA polymerases(including reverse transcriptases) that are known require complexing ofan oligonucleotide to a single-stranded template (“priming”) to initiateDNA synthesis, whereas RNA replication and transcription (copying of RNAfrom DNA) generally do not require a primer. By its very nature of beingextended by a DNA polymerase, a priming oligonucleotide does notcomprise a 3′-blocking moiety.

As used herein, the term “amplification oligonucleotide complex” refersto two amplification oligonucleotides directly or indirectly joinedtogether, as discussed below. Thus, an amplification oligonucleotidecomplex is made of a first amplification oligonucleotide and a secondamplification oligonucleotide that are joined together.

A “tagged oligonucleotide” as used herein refers to an oligonucleotidethat comprises at least a first region and a second region, where thefirst region comprises a “target hybridizing sequence” which hybridizesa target nucleic acid sequence of interest, and where the second regioncomprises a “tag sequence” situated 5′ to the target hybridizingsequence and which does not stably hybridize or bind to a target nucleicacid containing the target nucleic acid sequence. Hybridization of thetarget hybridizing sequence to the target nucleic acid sequence producesa “tagged target nucleic acid sequence.” The features and designconsiderations for the target hybridizing sequence component would bethe same as for the priming oligonucleotides discussed herein. The “tagsequence” or “heterologous tag sequence” may be essentially any sequenceprovided that it does not stably hybridize to the target nucleic acidsequence of interest and, thereby, participate in detectableamplification of the native target (i.e., as would be found in abiological sample) prior to any sequence modification. The tag sequencepreferably does not stably hybridize to any sequence derived from thegenome of an organism being tested or, more particularly, to any targetnucleic acid under reaction conditions. A tag sequence that is presentin a tagged oligonucleotide is preferably designed so as not tosubstantially impair or interfere with the ability of the targethybridizing sequence to hybridize to its target sequence. Moreover, thetag sequence will be of sufficient length and composition such that oncea complement of the tag sequence has been incorporated into an initialDNA primer extension product, a tag-specific priming oligonucleotide canthen be used to participate in subsequent rounds of amplification asdescribed herein. A tag sequence of the present invention is typicallyat least 10 nucleotides in length, and may extend up to 15, 20, 25, 30,35, 40, 50 or more nucleotides in length. Skilled artisans willrecognize that the design of tag sequences and tagged oligonucleotidesfor use in the present invention can follow any of a number of suitablestrategies, while still achieving the objectives and advantagesdescribed herein. In certain embodiments, the tagged oligonucleotide isa “tagged priming oligonucleotide” comprising a tag sequence and atarget hybridizing sequence. In other embodiments, the taggedoligonucleotide is a “tagged promoter oligonucleotide” comprising a tagsequence, a target hybridizing sequence and a promoter sequence situated5′ to the tag sequence and effective for initiating transcriptiontherefrom. Exemplary tag sequences and methods of identifyingparticularly useful tag sequences are disclosed in commonly owned U.S.provisional patent application having Ser. No. 61/451,285. Thedisclosure of this provisional patent application is incorporated byreference.

Oligonucleotides that are not extended enzymatically include promoterprovider oligonucleotides and blocker oligonucleotides. Theseoligonucleotides hybridize to a target nucleic acid, or its complement,but are not extended in a template-directed manner by enzymaticpolymerase activity. To prevent enzymatic extension of anoligonucleotide, the 3′-terminus of the oligonucleotide can bechemically or structurally blocked using a blocking moiety, as isgenerally known in the art. Blocked oligonucleotides are described in,e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,824,518, and 7,374,885. Ablocked oligonucleotide refers to an oligonucleotide that includes achemical and/or structural modification that is usually near or at the3′ terminus and that prevents or impedes initiation of DNA synthesisfrom the oligonucleotide by enzymatic means. Examples of suchmodifications include use of a 3′2′-dideoxynucleotide base, a 3′non-nucleotide moiety that prevents enzymatic extension, or attachmentof a short sequence in 3′ to 5′ orientation to the oligonucleotide tomake a final oligonucleotide with two 5′ termini (i.e., a first 5′ to 3′oligonucleotide attached to a second, usually shorter, 5′ to 3′oligonucleotide by covalently joining the oligonucleotides at their 3′termini). Another example of a modification is a “cap” made up of asequence that is complementary to at least 3 nt at the 3′-end of theoligonucleotide such that the 5′-terminal base of the cap iscomplementary to the 3′-terminal base of the oligonucleotide. Althoughblocked oligonucleotides are not extended enzymatically, they mayparticipate in nucleic acid amplification, for example by hybridizing toa specific location on a nucleic acid template strand to impedesynthesis of a complementary strand beyond the position at which theblocked oligonucleotide is bound.

Sizes of the amplification oligonucleotides are generally determined bythe function portions that are included in the oligonucleotide.Component portions of a promoter primer or promoter provideroligonucleotide include a promoter sequence specific for an RNApolymerase (RNP). RNP and their corresponding promoter sequences arewell known and may be purified from or made synthetically in vitro byusing materials derived from a variety of sources, e.g., viruses,bacteriophages, fungi, yeast, bacteria, animal, plant or human cells.Examples of RNP and promoters include RNA polymerase III and itspromoter (U.S. Pat. No. 7,241,618), bacteriophage T7 RNA polymerase andits promoter or mutants thereof (U.S. Pat. Nos. 7,229,765 and7,078,170), RNA polymerase and promoter from Thermus thermophilus (U.S.Pat. No. 7,186,525), RNA polymerases from HIV-1 or HCV, and plantdirected RNPs (U.S. Pat. No. 7,060,813). A promoter primer or provideroligonucleotide includes a promoter sequence that is linked functionallyto the chosen RNP. Preferred embodiments of promoter primer or promoterprovider oligonucleotides include a T7 promoter sequence that is usedwith T7 RNP, where the promoter sequence is in the range of 25 to 30 nt,such as a promoter sequence of SEQ ID NO:1 (AATTTAATACGACTCACTATAGGGAGA)or SEQ ID NO:2 (GAAATTAATACGACTCACTATAGGGAGA).

Amplification oligonucleotides that include a tag portion typicallyinclude a tag sequence in a range of 5 to 40 nt, with preferredembodiments in a range of 10 to 25 nt, or 10 to 30 nt, or 15 to 30 nt.Amplification oligonucleotides that include a target specific (TS)portion typically include a TS sequence in a range of 10 to 45 nt, withpreferred embodiments in a range of 10 to 35 nt or 20 to 30 nt.Amplification oligonucleotides that include multiple tag sequencesand/or multiple TS sequences and/or a promoter sequence will be in asize range that is determined by the length of its individual functionalsequences. For example, a promoter primer or provider oligonucleotidethat includes a tag sequence and a TS sequence will be the sum of thesizes of the promoter, tag and TS sequences, and may optionally includelinking nucleotides or non-nucleotide portions (e.g., abasic linkers).Amplification oligonucleotides made up of multiple functional componentsas described herein may be covalently linked by standard phosphodiesterlinkages, nucleic acid analog linkages, or non-nucleic acid linkagesdirectly between the different functional portions or may benon-covalently linked together by using additional nucleic acidsequences or non-nucleic (e.g., abasic linkages) compounds that serve asspacers and/or linkages between functional portions. Some embodiments ofamplification oligonucleotides may be linked together to form a complexby using non-covalent linkages, such as by using interactions of bindingpair members between the oligonucleotides, which includes directhybridization of complementary sequences contained in two or moreoligonucleotides, or via a linking component (including one or moreadditional oligonucleotides) to which the individual binding pairmembers of an oligonucleotide complex bind.

As used herein, “amplification” of a target nucleic acid refers to theprocess of creating in vitro nucleic acid strands that are identical orcomplementary to at least a portion of a target nucleic acid sequence,or a universal or tag sequence that serves as a surrogate for the targetnucleic acid sequence, all of which are only made if the target nucleicacid is present in a sample. Typically, nucleic acid amplification usesone or more nucleic acid polymerase and/or transcriptase enzymes toproduce multiple copies of a target polynucleotide or fragments thereof,or of a sequence complementary to the target polynucleotide or fragmentsthereof, or of a universal or tag sequence that has been introduced intothe amplification system to serve as a surrogate for the targetpolynucleotide, such as in a detection step, to indicate the presence ofthe target polynucleotide at some point in the assay, or as a site forfurther priming in an amplification reaction, or for use in asequencing-related workflow or sequencing reaction. In vitro nucleicacid amplification techniques are well known and includetranscription-associated amplification methods, such asTranscription-Mediated Amplification (TMA) or Nucleic AcidSequence-Based Amplification (NASBA), and other methods such asPolymerase Chain Reaction (PCR), Reverse Transcriptase-PCR (RT-PCR),Replicase Mediated Amplification, and Ligase Chain Reaction (LCR).

As used herein, the term “linear amplification” refers to anamplification mechanism that is designed to produce an increase in thetarget nucleic acid linearly proportional to the amount of targetnucleic acid in the reaction. For instance, multiple RNA copies can bemade from a DNA target using a transcription-associated reaction, wherethe increase in the number of copies can be described by a linear factor(e.g., starting copies of template×100). In preferred embodiments, afirst phase linear amplification in a multiphase amplification procedureincreases the starting number of target nucleic acid strands or thecomplements thereof by at least 10 fold, more preferably 100 fold, orstill more preferably by 10 to 1,000 fold before the second phaseamplification reaction is begun. An example of a linear amplificationsystem is “T7-based Linear Amplification of DNA” (TLAD; see Liu et al.,BMC Genomics, 4: Art. No. 19, May 9, 2003). Other methods are disclosedherein. Accordingly, the term “linear amplification” refers to anamplification reaction which does not result in the exponentialamplification of a target nucleic acid sequence. The term “linearamplification” does not refer to a method that simply makes a singlecopy of a nucleic acid strand, such as the transcription of an RNAmolecule into a single cDNA molecule as in the case of reversetranscription (RT)-PCR.

As used herein, the term “exponential amplification” refers to nucleicacid amplification that is designed to produce an increase in the targetnucleic acid geometrically proportional to the amount of target nucleicacid in the reaction. For example, PCR produces one DNA strand for everyoriginal target strand and for every synthesized strand present.Similarly, transcription-associated amplification produces multiple RNAtranscripts for every original target strand and for every subsequentlysynthesized strand. The amplification is exponential because thesynthesized strands are used as templates in subsequent rounds ofamplification. An amplification reaction need not actually produceexponentially increasing amounts of nucleic acid to be consideredexponential amplification, so long as the amplification reaction isdesigned to produce such increases.

As used herein, the term “substantially isothermal amplification” refersto an amplification reaction that is conducted at a substantiallyconstant temperature. The isothermal portion of the reaction may bepreceded or followed by one or more steps at a variable temperature, forexample, a first denaturation step and a final heat inactivation step orcooling step. It will be understood that this definition by no meansexcludes certain, preferably small, variations in temperature but israther used to differentiate the isothermal amplification techniquesfrom other amplification techniques known in the art that basically relyon “cycling temperatures” in order to generate the amplified products.Isothermal amplification differs from PCR, for example, in that thelatter relies on cycles of denaturation by heating followed by primerhybridization and polymerization at a lower temperature.

Preferred embodiments of the disclosed methods use aspects of isothermalamplification systems that are generally referred to as“transcription-associated amplification” methods, which amplify a targetsequence by producing multiple transcripts from a nucleic acid template.Such methods generally use one or more oligonucleotides, of which oneprovides a promoter sequence, deoxyribonucleoside triphosphates (dNTPs),ribonucleoside triphosphates (rNTPs), and enzymes with RNA polymeraseand DNA polymerase activities to generate a functional promoter sequencenear the target sequence and then transcribe the target sequence fromthe promoter (e.g., U.S. Pat. Nos. 4,868,105, 5,124,246, 5,130,238,5,399,491, 5,437,990, 5,554,516 and 7,374,885; and PCT Pub. Nos. WO1988/001302, WO 1988/010315 and WO 1995/003430). Examples includeTranscription-Mediated Amplification (TMA), nucleic acid sequence basedamplification (NASBA) and Self-Sustained Sequence Replication (3SR).Although the disclosed preferred embodiments rely on TMA (U.S. Pat. Nos.5,399,491 and 5,554,516) or one-primer transcription-associatedamplification (U.S. Pat. Nos. 7,374,885, 7,696,337 and 7,939,260), aperson of ordinary skill in the art will appreciate that alternativeamplification methods based on polymerase mediated extension ofoligonucleotide sequences may also be used with the compositions and/ormethod steps described herein.

To aid in understanding of some of the embodiments disclosed herein, theTMA method that has been described in detail previously (e.g., U.S. Pat.Nos. 5,399,491, 5,554,516 and 5,824,518) is briefly summarized. In TMA,a target nucleic acid that contains the sequence to be amplified isprovided as single stranded nucleic acid (e.g., ssRNA or ssDNA). Anyconventional method of converting a double stranded nucleic acid (e.g.,dsDNA) to a single-stranded nucleic acid may be used. A promoter primerbinds specifically to the target nucleic acid at its target sequence anda reverse transcriptase (RT) extends the 3′ end of the promoter primerusing the target strand as a template to create a cDNA copy, resultingin a RNA:cDNA duplex. RNase activity (e.g., RNase H of RT enzyme)digests the RNA of the RNA:cDNA duplex and a second primer bindsspecifically to its target sequence in the cDNA, downstream from thepromoter-primer end. Then RT synthesizes a new DNA strand by extendingthe 3′ end of the second primer using the cDNA as a template to create adsDNA that contains a functional promoter sequence. RNA polymerasespecific for the functional promoter initiates transcription to produceabout 100 to 1000 RNA transcripts (amplified copies or amplicons)complementary to the initial target strand. The second primer bindsspecifically to its target sequence in each amplicon and RT creates acDNA from the amplicon RNA template to produce a RNA:cDNA duplex. RNasedigests the amplicon RNA from the RNA:cDNA duplex and thetarget-specific sequence of the promoter primer binds to itscomplementary sequence in the newly synthesized DNA and RT extends the3′ end of the promoter primer as well as the 3′ end of the cDNA tocreate a dsDNA that contains a functional promoter to which the RNApolymerase binds and transcribes additional amplicons that arecomplementary to the target strand. Autocatalytic cycles that use thesesteps repeatedly during the reaction produce about a billion-foldamplification of the initial target sequence. Amplicons may be detectedduring amplification (real-time detection) or at an end point of thereaction (end-point detection) by using a probe that binds specificallyto a sequence contained in the amplicons. Detection of a signalresulting from the bound probes indicates the presence of the targetnucleic acid in the sample.

Another form of transcription associated amplification that uses asingle primer and one or more additional amplification oligonucleotidesto amplify nucleic acids in vitro by making transcripts that indicatethe presence of the target nucleic acid has been described in detailpreviously (U.S. Pat. Nos. 7,374,885, 7,696,337 and 7,939,260). Briefly,this single-primer method uses a priming oligonucleotide, a promoteroligonucleotide (or promoter provider oligonucleotide) that is modifiedto prevent the initiation of DNA synthesis from its 3′ end and,optionally, a blocker molecule (e.g., a 3′-blocked oligonucleotide) toterminate elongation of a cDNA from the target strand. The methodsynthesizes multiple copies of a target sequence by treating a targetnucleic acid that includes a RNA target sequence with (i) a primingoligonucleotide which hybridizes to the 3′-end of the target sequencesuch that a primer extension reaction can be initiated therefrom and(ii) a blocker molecule that binds to the target nucleic acid adjacentto or near the 5′-end of the target sequence. The primingoligonucleotide is extended in a primer extension reaction by using aDNA polymerase to give a DNA primer extension product complementary tothe target sequence, in which the DNA primer extension product has a 3′end determined by the blocker molecule and which is complementary to the5′-end of the target sequence. The method then separates the DNA primerextension product from the target sequence by using an enzyme whichselectively degrades the target sequence and treats the DNA primerextension product with a promoter oligonucleotide made up of a firstregion that hybridizes to a 3′-region of the DNA primer extensionproduct to form a promoter oligonucleotide:DNA primer extension producthybrid, and a second region that is a promoter for an RNA polymerasewhich is situated 5′ to the first region, where the promoteroligonucleotide is modified to prevent the initiation of DNA synthesisfrom the promoter oligonucleotide. The method extends the 3′-end of theDNA primer extension product in the promoter oligonucleotide:DNA primerextension product hybrid to add a sequence complementary to the secondregion of the promoter oligonucleotide, which is used to transcribemultiple RNA products complementary to the DNA primer extension productusing an RNA polymerase which recognizes the promoter and initiatestranscription therefrom. This method produces RNA transcripts that aresubstantially identical to the target sequence.

An embodiment of the one-primer Transcription-Mediated Amplificationmethod synthesizes multiple copies of an RNA target sequence byhybridizing to the target RNA a primer at a location in the 3′ portionof the target sequence and a 3′ blocked oligonucleotide (i.e., theblocker oligonucleotide) at a location in the 5′ portion of the targetsequence. Then the DNA polymerase activity of RT initiates extensionsfrom the 3′ end of the primer to produce a cDNA in a duplex with thetemplate strand (a RNA:cDNA duplex). The 3′ blocked oligonucleotidebinds to the target strand at a position adjacent to the intended 5′ endof the sequence to be amplified because the bound 3′ blockedoligonucleotide impedes extension of the cDNA beyond that location. Thatis, the 3′ end of the cDNA is determined by the position of the blockermolecule because polymerization stops when the extension product reachesthe blocking molecule bound to the target strand. The RNA:cDNA duplex isseparated by RNAse activity (RNase H of RT) that degrades the RNA,although those skilled in the art will appreciate that any form ofstrand separation may be used. A promoter provider oligonucleotideincludes a 5′ promoter sequence for an RNA polymerase and a 3′ sequencecomplementary to a sequence in the 3′ region of the cDNA to which ithybridizes. The promoter provider oligonucleotide has a modified 3′ endthat includes a blocking moiety to prevent initiation of DNA synthesisfrom the 3′ end of the promoter provider oligonucleotide. In the duplexmade of the promoter provider hybridized to the cDNA, the 3′-end of thecDNA is extended by using DNA polymerase activity of RT and the promoterprovider oligonucleotide serves as a template to add a promoter sequenceto the 3′ end of the cDNA, which creates a functional double-strandedpromoter made up of the sequence on the promoter provideroligonucleotide and the complementary cDNA sequence made from thepromoter provider template. RNA polymerase specific for the promotersequence binds to the functional promoter and transcribes multiple RNAtranscripts that are complementary to the cDNA and substantiallyidentical to the target sequence of the initial target RNA strand. Theresulting amplified RNA can cycle through the process again by bindingthe primer and serving as a template for further cDNA production,ultimately producing many amplicons from the initial target nucleic acidpresent in the sample. Embodiments of the single primer transcriptionassociated amplification method do not require use of the 3′ blockedoligonucleotide that serves as a blocker molecule and, if a bindingmolecule is not included the cDNA product made from the primer has anindeterminate 3′ end, but amplification proceeds substantially the sameas described above. Due to the nature of this amplification method, itis performed under substantially isothermal conditions, i.e., withoutcycles of raising and lowering incubation temperatures to separatestrands or allow hybridization of primers as is used in PCR-basedmethods.

As used herein, the term “tag” refers to a nucleotide sequencecovalently attached to a target-specific sequence of an oligonucleotidefor the purpose of conferring some additional functionality beyondbinding to the target sequence. Non-limiting examples of oligonucleotidetags include a 5′ promoter for an RNA polymerase, a primer binding site,a sequencing tag, a mass tag, a bar code tag, a capture tag, and soforth (e.g., U.S. Pat. Nos. 5,422,252, 5,882,856, 6,828,098, and PCTPub. No. 05/019479). An oligonucleotide tag can be unique to each targetsequence or universal (shared by a plurality of target sequences, e.g.,U.S. Pat. No. 5,104,792), depending on the specifics of a particularassay.

As used herein, “detection” of the amplified products may beaccomplished by using any known method. For example, the amplifiednucleic acids may be associated with a surface that results in adetectable physical change (e.g., an electrical change). Amplifiednucleic acids may be detected in solution phase or by concentrating themin or on a matrix and detecting labels associated with them (e.g., anintercalating agent such as ethidium bromide or cyber green). Otherdetection methods use probes complementary to a sequence in theamplified product and detect the presence of the probe:product complex,or use a complex of probes to amplify the signal detected from amplifiedproducts (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503 and 5,849,481).Other detection methods use a probe in which signal production is linkedto the presence of the target sequence because a change in signalresults only when the labeled probe binds to amplified product, such asin a molecular beacon, molecular torch, or hybridization switch probe(e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097,6,361,945, 6,534,274, 6,835,542, 6,849,412 and 8,034,554; and U.S. Pub.No. 2006/0194240 A1). Such probes typically use a label (e.g.,fluorophore) attached to one end of the probe and an interactingcompound (e.g., quencher) attached to another location of the probe toinhibit signal production from the label when the probe is in oneconformation (“closed”) that indicates it is not hybridized to amplifiedproduct, but a detectable signal is produced when the probe ishybridized to the amplified product which changes its conformation (to“open”). Detection of a signal from directly or indirectly labeledprobes that specifically associate with the amplified product indicatesthe presence of the target nucleic acid that was amplified.

As used herein, the term “label” refers to a molecular moiety orcompound that can be detected or lead to a detectable response, whichmay be joined directly or indirectly to a nucleic acid probe. Directlabeling may use bonds or interactions to link label and probe, whichincludes covalent bonds, non-covalent interactions (hydrogen bonds,hydrophobic and ionic interactions), or chelates or coordinationcomplexes. Indirect labeling may use a bridging moiety or linker (e.g.antibody, oligonucleotide, or other compound), which is directly orindirectly labeled, which may amplify a signal. Labels include anydetectable moiety. Examples of useful detectable moieties includeradionuclides, ligands such as biotin or avidin, enzymes, enzymesubstrates, reactive groups, chromophores (detectable dyes, particles,or beads), fluorophores, or luminescent compounds (e.g., bioluminescent,phosphorescent, or chemiluminescent label). Preferred chemiluminescentlabels include acridinium ester (“AE”) and derivatives thereof (U.S.Pat. Nos. 5,639,604, 5,656,207 and 5,658,737). Preferred labels aredetectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change compared to that of unbound labeledprobe, e.g., stability or differential degradation, without requiringphysical separation of bound from unbound forms (e.g., U.S. Pat. Nos.5,283,174, 5,656,207 and 5,658,737). Methods of synthesizing labels,attaching labels to nucleic acids, and detecting labels are well known(e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),Chapter 10; U.S. Pat. Nos. 4,581,333, 5,283,174, 5,547,842, 5,656,207and 5,658,737).

Members of a specific binding pair (or binding partners) are moietiesthat specifically recognize and bind to each other. Members may bereferred to as a first binding pair member (BPM1) and second bindingpair member (BPM2), which represent a variety of moieties thatspecifically bind together. Specific binding pairs are exemplified by areceptor and its ligand, enzyme and its substrate, cofactor or coenzyme,an antibody or Fab fragment and its antigen or ligand, a sugar andlectin, biotin and streptavidin or avidin, a ligand and chelating agent,a protein or amino acid and its specific binding metal such as histidineand nickel, substantially complementary polynucleotide sequences, whichinclude completely or partially complementary sequences, andcomplementary homopolymeric sequences. Specific binding pairs may benaturally occurring (e.g., enzyme and substrate), synthetic (e.g.,synthetic receptor and synthetic ligand), or a combination of anaturally occurring BPM and a synthetic BPM.

As used herein, the term “target capture” refers to selectivelyseparating a target nucleic acid from other components of a samplemixture, such as cellular fragments, organelles, proteins, lipids,carbohydrates, or other nucleic acids. A target capture system may bespecific and selectively separate a predetermined target nucleic acidfrom other sample components (e.g., by using a sequence specific to theintended target nucleic acid), or it may be nonspecific and selectivelyseparate a target nucleic acid from other sample components by usingother characteristics of the target (e.g., a physical trait of thetarget nucleic acid that distinguishes it from other sample componentswhich do not exhibit that physical characteristic). Preferred targetcapture methods and compositions have been previously described indetail (U.S. Pat. Nos. 6,110,678 and 6,534,273; and US Pub. No.2008/0286775 A1). Preferred target capture embodiments use a targetcapture oligonucleotide in solution phase and an immobilized captureprobe attached to a support to form a complex with the target nucleicacid and separate the captured target from other components.

As used herein, the term “target capture oligonucleotide” refers to atleast one nucleic acid oligonucleotide that bridges or joins a targetnucleic acid and an immobilized capture probe by using binding pairmembers, such as complementary nucleic acid sequences or biotin andstreptavidin. In one approach, the target capture oligonucleotide bindsnonspecifically to the target nucleic acid and immobilizes it to a solidsupport. In a different approach, a target specific (TS) sequence of thetarget capture oligonucleotide binds specifically to a sequence in thetarget nucleic acid. In both approaches the target captureoligonucleotide includes an immobilized capture probe-binding regionthat binds to an immobilized capture probe (e.g., by specific bindingpair interaction). In embodiments in which the TS sequence and theimmobilized capture probe-binding region are both nucleic acidsequences, they may be covalently joined to each other, or may be ondifferent oligonucleotides joined by one or more linkers.

An “immobilized capture probe” provides a means for joining a targetcapture oligonucleotide to a solid support. The immobilized captureprobe is a base sequence recognition molecule joined to the solidsupport, which facilitates separation of bound target polynucleotidefrom unbound material. Any known solid support may be used, such asmatrices and particles free in solution. For example, solid supports maybe nitrocellulose, nylon, glass, polyacrylate, mixed polymers,polystyrene, silane polypropylene and, preferably, magneticallyattractable particles. Particularly preferred supports include magneticspheres that are monodisperse (i.e., uniform in size±about 5%), therebyproviding consistent results, which is particularly advantageous for usein an automated assay. The immobilized capture probe may be joineddirectly (e.g., via a covalent linkage or ionic interaction), orindirectly to the solid support. Common examples of useful solidsupports include magnetic particles or beads.

As used herein, the term “separating” or “purifying” generally refers toremoval of one or more components of a mixture, such as a sample, fromone or more other components in the mixture. Sample components includenucleic acids in a generally aqueous solution phase, which may includecellular fragments, proteins, carbohydrates, lipids, and othercompounds. Preferred embodiments separate or remove at least 70% to 80%,and more preferably about 95%, of the target nucleic acid from othercomponents in the mixture.

B. Methods of Multiphase Amplification

As noted above, the first aspect of the present invention is concernedwith a method for amplifying a target nucleic acid sequence in a sampleincluding the following steps. Initially, the target nucleic acidsequence is subjected to a first phase amplification reaction underconditions that do not support exponential amplification of the targetnucleic acid sequence. The first phase amplification reaction generatesa first amplification product, which is subsequently subjected to asecond phase amplification reaction under conditions allowingexponential amplification of the first amplification product, therebygenerating a second amplification product.

In this aspect, the target nucleic acid sequence may be any RNA or DNAsequence; however, in preferred embodiments, the target sequence is anRNA sequence. In some embodiments, before the first amplification step,the sample may be contacted with a first amplification oligonucleotideunder conditions allowing hybridization of the first amplificationoligonucleotide to a portion of the target nucleic acid sequence in thesample. The first amplification oligonucleotide usually includes atarget specific sequence and optionally, one or more tag sequences. Inpreferred embodiments, the tag sequence may be a 5′ promoter sequencerecognized by an RNA polymerase, such as T7 RNA polymerase, anamplification primer binding site, a specific binding site used forcapture, or a sequencing primer binding site. In some embodiments, asecond amplification oligonucleotide may be used in combination with thefirst amplification oligonucleotide before the first amplification step.

In many cases, it may be desirable to isolate the target nucleic acidsequence prior to the first phase amplification. To this end, the samplemay be contacted with a target capture oligonucleotide under conditionsallowing hybridization of the target capture oligonucleotide to aportion of the target nucleic acid sequence. In some embodiments, thetarget nucleic acid is captured onto the solid support directly, forexample by interaction with an immobilized capture probe. Alternatively,the target nucleic acid is captured onto the solid support as a memberof a three molecule complex, with the target capture oligonucleotidebridging the target nucleic acid and the immobilized capture probe. Ineither scenario, the solid support typically includes a plurality ofmagnetic or magnetizable particles or beads that can be manipulatedusing a magnetic field. Preferably, the step of isolating the targetnucleic acid sequence also includes washing the target captureoligonucleotide:target nucleic acid sequence hybrid to remove undesiredcomponents that may interfere with subsequent amplification.

The step of isolating the target nucleic acid sequence may sometimesinclude contacting the sample with a first amplification oligonucleotideunder conditions allowing hybridization of the first amplificationoligonucleotide to a portion of the target nucleic acid sequence. Insome embodiments, the portion of the target sequence targeted by thefirst amplification oligonucleotide may be completely different (e.g.non-overlapping) from the portion targeted by the target captureoligonucleotide. Alternatively, the portion of the target sequencetargeted by the first amplification oligonucleotide may fully orpartially overlap with, or even be identical to, the portion targeted bythe target capture oligonucleotide. The first amplificationoligonucleotide usually includes a target specific sequence andoptionally, one or more tag sequence(s). In preferred embodiments, thetag sequence may be a 5′ promoter sequence recognized by an RNApolymerase, such as T7 RNA polymerase, and other functional sites asdescribed above. In some embodiments, a second amplificationoligonucleotide may be used in combination with the first amplificationoligonucleotide during the target nucleic acid sequence isolation step.It is contemplated that the first and second amplificationoligonucleotides may form a complex, e.g., a direct hybrid (DH) complex.In some embodiments, at least one of the first and second amplificationoligonucleotides may include a tag sequence (e.g., a universal tag)located 5′ to a target specific sequence, which tag sequence may betargeted by an amplification oligonucleotide during the second phaseamplification.

The amplification oligonucleotide primers are often provided in a targetcapture reagent. In certain preferred embodiments, the target capturereagent includes only one of the amplification oligonucleotides to beused in the production of a particular amplification product in a firstphase amplification reaction. The amplification oligonucleotides can behybridized to a target nucleic acid, and isolated along with the targetsequence during the target capture step. One advantage of this method isthat by hybridizing the amplification oligonucleotide to the targetnucleic acid during target capture, the captured nucleic acids can bewashed to remove sample components, such as unhybridized amplificationoligonucleotide primers, providers, and/or complexes. In a multiplexreaction, removing unhybridized amplification oligonucleotides allowsthe multiplex amplification reaction to occur without interference fromthe excess amplification oligonucleotides, thereby substantiallyreducing or eliminating the problems common to multiplex reactions.Further, if the amplification oligonucleotide or amplificationoligonucleotide complex comprises a tag sequence, then the tag isincorporated into the first amplification product, thereby allowing forsubsequent amplification using primers specific for the tag sequence.

As noted above, the first phase amplification reaction is carried outunder conditions that do not support exponential amplification of thetarget nucleic acid sequence. In preferred embodiments, the first phaseamplification reaction is a linear amplification reaction. The firstphase amplification reaction will typically produce from about 2-fold toabout 10,000-fold amplification, and preferably from about 10-fold toabout 10,000-fold amplification, of the target nucleic acid sequence. Insome embodiments, the first phase amplification reaction issubstantially isothermal, i.e., it does not involve thermal cyclingcharacteristic of PCR and other popular amplification techniques.Typically, the first phase amplification reaction will involvecontacting the target nucleic acid sequence with a first phaseamplification reaction mixture that supports linear amplification of thetarget nucleic acid sequence and lacks at least one component that isrequired for its exponential amplification. In some embodiments, thefirst phase amplification reaction mixture includes an amplificationenzyme selected from a reverse transcriptase, a polymerase, and acombination thereof. The polymerase is typically selected from anRNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aDNA-dependent RNA polymerase, and a combination thereof. In preferredembodiments, the first phase amplification reaction mixture furtherincludes a ribonuclease (RNase), such as an RNase H or a reversetranscriptase with an RNase H activity. Preferably, the first phaseamplification mixture includes a reverse transcriptase with an RNase Hactivity and an RNA polymerase.

In some embodiments, the first phase amplification mixture may alsoinclude an amplification oligonucleotide. Preferably, the amplificationoligonucleotide includes a 5′ promoter sequence for an RNA polymerase,such as T7 RNA polymerase, and/or a blocked 3′ terminus that preventsits enzymatic extension. In addition, the first phase amplificationmixture may sometimes include a blocker oligonucleotide to preventenzymatic extension of the target nucleic sequence beyond a desiredend-point.

As noted above, the key feature of the first phase amplificationreaction is its inability to support an exponential amplificationreaction because one or more components required for exponentialamplification are lacking, and/or an agent is present which inhibitsexponential amplification, and/or the temperature of the reactionmixture is not conducive to exponential amplification, etc. Withoutlimitation, the lacking component required for exponential amplificationand/or inhibitor and/or reaction condition may be selected from thefollowing group: an amplification oligonucleotide (e.g., anamplification oligonucleotide comprising a 5′ promoter sequence for anRNA polymerase, a non-promoter amplification oligonucleotide, or acombination thereof), an enzyme (e.g., a polymerase, such as an RNApolymerase), a nuclease (e.g., an exonuclease, an endonuclease, acleavase, an RNase, a phosphorylase, a glycosylase, etc), an enzymeco-factor, a chelator (e.g., EDTA or EGTA), ribonucleotide triphosphates(rNTPs), deoxyribonucleotide triphosphates (dNTPs), Mg²⁺, a salt, abuffer, an enzyme inhibitor, a blocking oligonucleotide, pH,temperature, salt concentration and a combination thereof. In somecases, the lacking component may be involved indirectly, such as anagent that reverses the effects of an inhibitor of exponentialamplification which is present in the first phase reaction.

As noted above, the second phase (or later, if there are more than 2phases) amplification reaction is carried out under conditions thatallow exponential amplification of the target nucleic acid sequence.Therefore, in preferred embodiments, the second phase amplificationreaction is an exponential amplification reaction. Much like the firstphase amplification reaction, the second phase amplification reaction ispreferably a substantially isothermal reaction, such as, for example, atranscription-associated amplification reaction or a strand displacementamplification reaction. In particularly preferred embodiments, thesecond phase amplification reaction is a Transcription-MediatedAmplification (TMA) reaction.

The second (or later) phase amplification usually involves contactingthe first amplification product with a second phase amplificationreaction mixture which, in combination with the first phaseamplification reaction mixture, will support exponential amplificationof the target nucleic acid sequence. Thus, the second phaseamplification reaction mixture typically includes, at a minimum, the oneor more component(s) required for exponential amplification that thefirst phase amplification reaction mixture is lacking. In someembodiments, the second phase amplification reaction mixture includes acomponent selected from an amplification oligonucleotide, a reversetranscriptase, a polymerase, a nuclease, a phosphorylase, an enzymeco-factor, a chelator, ribonucleotide triphosphates (rNTPs),deoxyribonucleotide triphosphates (dNTPs), Mg²⁺, an optimal pH, anoptimal temperature, a salt and a combination thereof. The polymerase istypically selected from an RNA-dependent DNA polymerase, a DNA-dependentDNA polymerase, a DNA-dependent RNA polymerase, and a combinationthereof. In some embodiments, the second phase amplification reactionmixture further includes an RNase, such as an RNase H or a reversetranscriptase with an RNase H activity. In some cases, the second phaseamplification reaction mixture includes an amplificationoligonucleotide, a reverse transcriptase with an RNase H activity, andan RNA polymerase.

The method of the present invention may be used to quantify a targetnucleic acid sequence in a biological sample. To this end, the secondphase amplification reaction is preferably a quantitative amplificationreaction. Typically, the present method will include an additional stepof detecting the second amplification product using a variety ofdetection techniques, e.g., a detection probe, a sequencing reaction,electrophoresis, mass spectroscopy, melt curve analysis, or acombination thereof. Preferably, the second amplification product isquantified using a detection probe. In some embodiments, thequantification step may be performed in real time, which can beaccomplished, for example, if the detection probe used for thequantification is a molecular beacon, a molecular torch, a hybridizationswitch probe, or a combination thereof. The detection probe may beincluded in the first and/or second phase amplification reactions withsubstantially equal degree of success.

In some embodiments, the present method further includes a step ofcontacting the second amplification product with another bolus of anamplification component selected from an amplification oligonucleotide,a reverse transcriptase (e.g., a reverse transcriptase with an RNase Hactivity), a polymerase (e.g., an RNA polymerase), a nuclease, aphosphorylase, an enzyme co-factor, a chelator, ribonucleotidetriphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs), Mg²⁺,a salt and a combination thereof. The purpose of this additional step isto provide a boost to the second phase amplification reaction as some ofthe amplification reaction components may become depleted over time.

A closely related aspect of the present invention is concerned with amethod for amplifying a plurality of different target nucleic acidsequences in a sample including the following steps. Initially, thetarget nucleic acid sequences are subjected to a first phaseamplification reaction under conditions that do not support exponentialamplification of any of the target nucleic acid sequences. The firstphase amplification reaction generates a plurality of firstamplification products, which are subsequently subjected to a second (orlater) phase amplification reaction under conditions allowingexponential amplification of the first amplification products, therebygenerating a plurality of second amplification products.

In a modified version of the second aspect, the invention provides amethod for amplifying a plurality of different target nucleic acidsequences in a sample, where some, but not all, of the target nucleicacid sequences are subjected to linear amplification, and/or some, butnot all, of the target nucleic acid sequences are subjected toexponential amplification. At least three variants of the first phaseamplification are contemplated: (1) some of the target sequences aresubjected to linear amplification, and the rest are left unamplified;(2) some of the target sequences are subjected to exponentialamplification, and the rest are left unamplified; and (3) some of thetarget sequences are subjected to linear amplification, and the rest aresubjected to exponential amplification. Thus, the first phaseamplification may result in amplification of all of the target nucleicacid sequences (option 3) or only a subset thereof (options 1 and 2).The subset of the target nucleic acid sequences may represent targetsknown to be present in relatively low quantities and/or targets that aredifficult to amplify compared to other targets. The first phaseamplification reaction generates one or more first amplificationproduct(s). The first amplification product(s) and any unamplifiedtarget nucleic acid sequence(s) in the sample are then subjected to asecond phase amplification reaction under conditions allowingexponential amplification thereof, generating a plurality of secondamplification products.

It is understood that the various optional elements and parametersdiscussed above in connection with multiphase uniplex (i.e. singletarget) amplification are also applicable to the multiphase multiplexamplification modes described herein.

C. Compositions for Multiphase Amplification

As noted above, in a third aspect, the present invention provides acomposition for amplifying a target nucleic acid sequence in a sampleincluding the following components: (a) an amplification oligonucleotidethat hybridizes to a first portion of the target nucleic acid sequence;(b) an optional target capture oligonucleotide that hybridizes to asecond portion of the target nucleic acid sequence; and (c) anamplification enzyme. One of the key features of the present compositionis that it lacks at least one component required for exponentialamplification of the target nucleic acid sequence. As explained indetail elsewhere in this application, one of the advantages of thepresent composition is that it helps to reduce non-specificamplification, thereby focusing the amplification resources on thetarget sequence.

In this aspect, the target nucleic acid sequence may be any RNA or DNAsequence. In some embodiments, the portion of the target sequencetargeted by the first amplification oligonucleotide may be completelydifferent (e.g. non-overlapping) from the portion targeted by the targetcapture oligonucleotide (if used). Alternatively, the portion of thetarget sequence targeted by the first amplification oligonucleotide mayfully or partially overlap with, or even be identical to, the portiontargeted by the target capture oligonucleotide. In some special cases,the target capture oligonucleotide may also be structurally identical tothe amplification oligonucleotide and perform an amplification functionin addition to target capture. The target capture oligonucleotide may bedirectly coupled to a solid support (e.g., via covalent bonding);alternatively, the composition may further include a capture probecoupled to a solid support such that the capture probe hybridizes to aportion of the target capture oligonucleotide. The solid supportpreferably includes a plurality of magnetic or magnetizable particles orbeads that can be manipulated using a magnetic field.

As noted above, the amplification oligonucleotide of the presentcomposition may include a target specific sequence and 5′ promotersequence recognized by an RNA polymerase, such as T7 RNA polymerase. Insome embodiments, the composition may include at least two amplificationoligonucleotides, one of which may include a 5′ promoter sequence for anRNA polymerase (e.g., T7 RNA polymerase). The promoter-containingamplification oligonucleotide may further include a blocked 3′ terminusthat prevents its enzymatic extension. In those cases where thecomposition includes two or more amplification oligonucleotides, theoligonucleotides may form a complex, e.g., a DH complex. The DH complexmay include a non-promoter amplification oligonucleotide that includes atarget specific sequence joined at its 5′ terminus to a linking memberfor linking the non-promoter amplification oligonucleotide to a secondamplification oligonucleotide of the DH complex. The secondamplification oligonucleotide typically includes a 5′ promoter sequencefor an RNA polymerase, such as T7 RNA polymerase. As explained in moredetail above in connection with single-primer amplification, the secondamplification oligonucleotide may sometimes include a blocked 3′terminus that prevents its enzymatic extension. The linking member ofthe non-promoter amplification oligonucleotide typically includes anucleotide sequence that is complementary to a portion of the secondamplification oligonucleotide. In cases where the second amplificationoligonucleotide includes a promoter sequence, the linking member of thefirst amplification oligonucleotide preferably includes a nucleotidesequence that is complementary to a portion of the promoter sequence ofthe second amplification oligonucleotide. In some embodiments, at leastone of the amplification oligonucleotides may include a tag sequence(e.g., a universal tag) located 5′ to a target specific sequence. Inaddition, the present composition may include a blocker oligonucleotideto prevent enzymatic extension of the target nucleic sequence beyond adesired end-point.

The amplification enzyme of the present composition may be in the formof a reverse transcriptase, a polymerase, or a combination thereof. Thepolymerase may be selected from an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, and acombination thereof. The composition preferably further includes anRNase, such as an RNase H or a reverse transcriptase with an RNase Hactivity. In some instances, the composition may further include adetection probe for monitoring the isothermal amplification reaction inreal time, which detection probe may be selected from a molecularbeacon, a molecular torch, a hybridization switch probe, and acombination thereof.

As noted above, one of the key features of the present composition isits lack of at least one component required for exponentialamplification of the target nucleic acid sequence. The lacking componentrequired for exponential amplification may be an amplificationoligonucleotide (e.g., a promoter primer or a non-promoter primer), apolymerase (e.g., an RNA polymerase), a nuclease, a phosphorylase, anenzyme co-factor, a chelator, one or more ribonucleotide triphosphates(rNTPs), Mg²⁺, an optimal pH, an optimal temperature, a salt, an optimalsalt concentration or a combination thereof. It is understood that thislist is not exhaustive and may include other components that arenecessary for an exponential amplification reaction to proceed.

Where multiplex amplification is intended, the present composition mayinclude a plurality of different target capture oligonucleotides and aplurality of different amplification oligonucleotides that hybridize toa plurality of different target nucleic acid sequences.

As noted above, the present invention also provides an alternativecomposition for amplifying a plurality of different target nucleic acidsequences in a sample. This alternative composition includes thefollowing components: (a) a plurality of different amplificationoligonucleotide complexes that hybridize to a plurality of differenttarget nucleic acid sequences, where each amplification oligonucleotidecomplex includes a first amplification oligonucleotide having a firsttarget specific sequence that is directly or indirectly joined to asecond amplification oligonucleotide having a second target specificsequence; and (b) an amplification enzyme. Once again, the compositionlacks at least one component required for exponential amplification ofthe target nucleic acid sequences.

One of the first and second amplification oligonucleotides typicallyincludes a 5′ promoter sequence for an RNA polymerase, such as T7 RNApolymerase. As explained in more detail above in connection withsingle-primer amplification, the promoter-containing amplificationoligonucleotide may include a blocked 3′ terminus that prevents itsenzymatic extension. In some embodiments, at least one of the first andsecond amplification oligonucleotides may also include a tag sequence(e.g., a universal tag) located 5′ to a target specific sequence. Thecomposition may further include a blocker oligonucleotide to preventenzymatic extension of the target nucleic sequence beyond a desiredend-point.

In some embodiments, the composition may also include a plurality ofdifferent target capture oligonucleotides that hybridize to the targetnucleic acid sequences. The target capture oligonucleotide may bedirectly coupled to a solid support (e.g., via covalent bonding);alternatively, the composition may further include a capture probecoupled to a solid support such that the capture probe hybridizes to aportion of the target capture oligonucleotide. The solid supportpreferably includes a plurality of magnetic or magnetizable particles orbeads that can be manipulated using a magnetic field.

As noted above, methods and compositions disclosed herein are useful foramplifying target nucleic acid sequences in vitro to produce amplifiedsequences that can be detected to indicate the presence of the targetnucleic acid in a sample. The methods and compositions are useful forsynthesizing amplified nucleic acids to provide useful information formaking diagnoses and/or prognoses of medical conditions, detecting thepurity or quality of environmental and/or food samples, or investigatingforensic evidence. The methods and compositions are advantageous becausethey allow synthesis of a variety of nucleic acids to provide highlysensitive assays over a wide dynamic range that are relatively rapid andinexpensive to perform, making them suitable for use in high throughputand/or automated systems. The methods and compositions can be used forassays that analyze single target sequences, i.e., uniplex amplificationsystems, and are especially useful for assays that simultaneouslyanalyze multiple different target sequences, i.e., multiplexamplification systems. Preferred compositions and reactions mixtures areprovided in kits that include defined assay components that are usefulbecause they allow a user to efficiently perform methods that use thecomponents together in an assay to amplify desired targets.

Embodiments of the compositions and methods described herein may befurther understood by the examples that follow. Method steps used in theexamples have been described herein and the following informationdescribes typical reagents and conditions used in the methods with moreparticularity. Those skilled in the art of nucleic acid amplificationwill appreciate that other reagents and conditions may be used that willnot substantially affecting the process or results so long as guidanceprovided in the description above is followed. For example, althoughTranscription-Mediated Amplification (TMA) methods are described in theexamples below, the claimed methods are not limited to TMA-basedembodiments. Moreover, those skilled in the art of molecular biologywill also understand that the disclosed methods and compositions may beperformed manually or in a system that performs one or more steps (e.g.,pipetting, mixing, incubation, and the like) in an automated device orused in any type of known device (e.g., test tubes, multi-tube unitdevices, multi-well devices such as 96-well microtitre plates, and thelike).

EXAMPLES

Exemplary reagents used in the methods described in the examples includethe following.

“Sample Transport Medium” or “STM” is a phosphate-buffered solution (pH6.7) that included EDTA, EGTA, and lithium lauryl sulfate (LLS).

“Target Capture Reagent” or “TCR” is a HEPES-buffered solution (pH 6.4)that included lithium chloride and EDTA, together with 250 μg/ml ofmagnetic particles (1 micron SERA-MAG™ MG-CM particles, Seradyn, Inc.Indianapolis, Ind.) with (dT)₁₄ oligonucleotides covalently boundthereto.

“Target Capture Wash Solution” or “TC Wash Solution” is a HEPES-bufferedsolution (pH 7.5) that included sodium chloride, EDTA, 0.3% (v/v)absolute ethanol, 0.02% (w/v) methyl paraben, 0.01% (w/v) propylparaben, and 0.1% (w/v) sodium lauryl sulfate.

“Amplification Reagent” or “AR” is a HEPES-buffered solution (pH 7.7)that included magnesium chloride, potassium chloride, fourdeoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), fourribonucleotide triphosphates (rATP), rCTP, rGTP, and rUTP). Primersand/or probes may be added to the reaction mixture in the amplificationreagent, or may be added separate from the reagent (primerlessamplification reagent).

“Enzyme Reagents” or “ER”, as used in amplification or pre-amplificationreaction mixtures, are HEPES-buffered solutions (pH 7.0) that includeMMLV reverse transcriptase (RT), T7 RNA polymerase, salts and cofactors.

Example 1 Standard Single-Phase Amplification Protocol

An exemplary protocol for standard single-phase TMA reactions thatdetect results in real time follows. The assay includes purification oftarget nucleic acids before amplification, amplification, and detectionof the amplified products during amplification.

Target capture is performed substantially as previously described indetail (U.S. Pat. Nos. 6,110,678, 6,280,952 and 6,534,273). Briefly,samples are prepared to contain known amounts of target RNA (in vitrotranscripts (“IVT”) present at a predetermined copy level per sample ina total volume of 400 ml of a 1:1 (v:v) mixture of water and sampletransport medium). Each sample is mixed with 100 ml of TCR thattypically contains 5 pmol of target capture oligonucleotide (TCO)specific for the analyte nucleic acid to be captured (i.e., 3′target-specific binding region) and a 5′ tail region (e.g., dT₃A₃₀sequence) for binding to the immobilized probe (e.g., poly-Toligonucleotides attached to paramagnetic particles; 12.5 μg ofparticles with attached oligonucleotides per reaction). The mixtures areincubated for 25 to 30 min at 60±1° C. and then for 25 to 30 min at roomtemperature (20 to 25° C.) to form hybridization complexes through whichtarget nucleic acids are bound to the paramagnetic particles isolatedvia magnetic separation (e.g., KingFisher96™ magnetic particleprocessor, Thermo Fisher Scientific, Inc., Waltham, Mass.) and washedone time using TC wash solution.

Particles are re-suspended in 0.075 ml of amplification reagent and withamplification oligonucleotides used in the amplification. Detectionprobes (e.g., molecular beacon or molecular torch probes labeled with afluorescent label compound) may be added with amplificationoligonucleotides, or with addition of enzymes, or following addition ofenzymes. Reaction mixtures are covered to prevent evaporation andincubated for 1 to 2 minutes at 42±0.5° C. While keeping them at 42±0.5°C., the mixtures were uncovered and mixed with 0.025 ml of enzymereagent per mixture, covered again, and incubated for 30 to 40 minutesat 42±0.5° C., during which time fluorescence was measured at regulartime intervals (e.g., every minute or several reads per minute) whichare referred to as “cycles” for data collection and display, which istypically a graph of detected fluorescence units versus time, from whicha time of emergence of signal is determined (“TTime,” i.e. the time atwhich fluorescence signal for a sample becomes positive over apredetermined background level).

Example 2 Evaluation of Dual-Phase HIV-1 Amplification in Forward TMAFormat

In this example, dual-phase forward TMA was evaluated using a humanimmunodeficiency virus 1 (HIV-1), subtype B target template containingthe pol region.

In the dual-phase amplification approach used here, which is brieflysummarized in FIG. 1, a T7 primer was hybridized to the target HIV-1sequence during target capture, followed by removal of excess T7 primer.The amplification process was divided into two distinct phases. Duringthe first phase, a non-T7 primer was introduced along with all of therequisite amplification, detection and enzyme reagents, with theexception of additional T7 primer. In the presence of reversetranscriptase, the T7 primer hybridized to the target was extended,creating a cDNA copy, and the target RNA template was degraded by thereverse transcriptase's RNase H activity. The non-T7 primer subsequentlyhybridized to the cDNA and was then extended, filling in the promoterregion of the T7 primer and creating an active, double-strandedtemplate. T7 polymerase then produced multiple RNA transcripts from thetemplate. The non-T7 primer subsequently hybridized to the RNAtranscripts and was extended, producing promoterless cDNA copies of thetarget RNA template. The RNA strands were degraded by RNase activity ofthe reverse transcriptase. Because no T7 primer was available in thephase 1 amplification mixture, the reaction could not proceed anyfurther. The second phase was then started with the addition of T7primer, thus initiating exponential amplification of the cDNA poolproduced in phase 1.

Results from the initial evaluation of the dual-phase approach are shownin FIGS. 3A-3C. FIG. 3A shows results of a single-phase amplificationexperiment that was modified to mimic the dual-phase format. Morespecifically, the T7 primer was added during the target capture step(allowing the standard 60° C. annealing step to be eliminated from theprotocol); no primers or Enzyme Reagent were added in the first phase;and non-T7 and T7 primers as well as Enzyme Reagent were added to thesecond phase for the initiation of exponential amplification. To addressthe concern that this modified protocol for the standard single-phasecontrol may have somewhat compromised its performance, we compared themodified single-phase forward protocol to the standard single-phaseforward TMA (FIGS. 2A-2B). As one can see from FIGS. 2A and 2B, the twoprotocols resulted in highly similar levels of precision and sensitivityof detection. In contrast, the dual-phase protocol yielded significantlyimproved sensitivity and precision at the low end of analyteconcentration (˜20 copies/r×n) compared with the standard single-phaseformat under these conditions (FIG. 3B). Notably, the dual-phase formatyielded superior performance both in terms of precision and shorterdetection time (FIG. 3C).

Example 3 Optimization of Dual-Phase HIV-1 Amplification Parameters

The first priority in the optimization process was to slow the emergencetimes and separate the individual target input levels to allow accurateand precise quantification, as well as reduce any putative interferencewith the non-T7 primer. This was accomplished by titrating down the T7primer concentration in the second phase (amount used in the dual-phasereaction depicted in FIG. 3B was 10 pmol/r×n). The assay was shown toretain 10 copies/r×n sensitivity and high precision with the lowestamount of T7 provider tested (1.0 pmol/r×n; FIGS. 4A-4D).

Likewise, the non-T7 primer was also titrated down (amount used in thedual-phase reaction depicted in FIG. 3B was 15 pmol/r×n) while keepingthe T7 primer constant at 1 pmol/r×n. A level of 10 pmol/r×n was foundto be sufficient for sensitive amplification without losing precision(FIG. 5A). At a level of 2 pmol/r×n of non-T7 primer, the precision at10 copies/r×n was not as good as with 10 pmol/r×n of non-T7 primer (FIG.3B), but the performance of the assay was still superior to that of thesingle-phase control (FIG. 3A).

Thus, the present inventors unexpectedly found that the dual-phaseamplification format allows substantially reducing primer concentrationsand still attaining superior performance compared to the single-phaseformat, while reducing side product formation and multiplexinterference.

The need for an additional bolus of enzyme in the second phase was alsoexamined (FIGS. 6A-6C). Restriction of enzyme addition to the firstphase resulted in a moderate improvement in precision at the low end ofanalyte concentrations tested (FIGS. 6B and 6C). Further, each copylevel emerged approximately five minutes later relative to thedual-phase format where the enzyme reagent was present in both phases.However, the previously observed improvement in sensitivity wasretained.

Example 4 Dual-Phase Amplification of HPV16

To determine whether the dual-phase amplification format has broadapplicability beyond HIV-1 detection, it was tested on the humanpapillomavirus subtype 16 (HPV16).

The dual-phase amplification protocol was essentially the same as theone described above in Example 2. Briefly, a T7 primer was hybridized tothe target HPV16 sequence during target capture, followed by removal ofexcess T7 primer. In the first phase of amplification, a non-T7 primerwas added along with all of the requisite amplification, detection andenzyme reagents, with the exception of additional T7 primer. After fiveminutes at 42° C., the T7 primer was added to the reaction mixture toinitiate the exponential amplification phase, which was also carried outat 42° C. with real-time detection. The single-phase control experimentwas carried out using the same primers and detection probe in thestandard single-phase forward TMA format as described in Example 1.

Results of the HPV16 amplification experiment are shown in FIGS. 7A-7C.As shown in FIG. 7A, the single-phase format was able to detect thetarget template down to ˜500 copies/mL (˜200 copies/r×n), whereas thedual-phase format improved the sensitivity over 15 fold to ˜30 copies/mL(˜13 copies/r×n) (FIG. 7B). Further, consistent with our priorobservations, the dual-phase amplification format was associated with asignificant reduction in the time of detection compared with thesingle-phase format (FIG. 7C).

Example 5 Dual-Phase Amplification of PCA3

In addition, the dual-phase amplification format was tested on theprostate cancer antigen 3 (PCA3).

A similar dual-phase amplification protocol was used. Briefly, a T7primer was hybridized to the target PCA3 sequence during target capture,followed by removal of excess T7 primer. In the first phase ofamplification, a non-T7 primer was added along with all of the requisiteamplification and enzyme reagents, with the exception of additional T7primer and a molecular torch detection probe. After five minutes at 42°C., the T7 primer and the detection probe were added to the reactionmixture to start the exponential amplification phase, which was alsocarried out at 42° C. with real-time detection. The single-phase controlexperiment was carried out using the same primers and detection probe inthe standard single-phase TMA format.

Results of the PCA3 amplification experiment are shown in FIGS. 8A-8C.As one can see from FIGS. 8A-8B, the dual-phase format yielded asignificantly improved sensitivity and precision at the low end ofanalyte concentration (˜130 copies/ml, which equivalent to ˜50copies/r×n) compared with the standard single-phase format. In addition,similar to our prior observations, the dual-phase amplification formatwas associated with a significant reduction in the time of detectioncompared with the single-phase format (FIG. 8C).

Example 6 Dual-Phase Co-Amplification of PCA3 and T2-ERG

Next, we employed the dual-phase amplification format for simultaneousamplification of multiple targets. In this example, PCA3 and T2-ERGtarget templates were co-amplified using the dual-phase forward TMAprotocol to determine whether duplex amplification will result in thesame improvement in sensitivity and precision we have observedpreviously in uniplex assays (Examples 2-5), as well as provide areduction in the interference between analytes which is often observedin a standard single phase format.

Briefly, T7 primers were hybridized to the target PCA3 and T2-ERGsequences during target capture, followed by removal of excess T7primers. In the first phase of amplification, non-T7 primers were addedalong with all of the requisite amplification, detection and enzymereagents, with the exception of additional T7 primers. After fiveminutes at 42° C., the T7 primers were added to the reaction mixture toinitiate the exponential amplification phase, which was also carried outat 42° C. with real-time detection in two different fluorescent channels(one for each target). The single-phase control experiment was carriedout using the same primers and detection probes in the standardsingle-phase forward TMA format as described in Example 1.

Results of the PCA3 amplification in the presence of T2-ERG are shown inFIGS. 9A-9C. As one can see from FIGS. 9A-9B, the dual-phase formatyielded a significantly improved sensitivity and precision at the lowend of analyte concentration (˜1,250 copies/ml, which is equivalent to˜500 copies/r×n) compared with the standard single-phase format. Theseresults demonstrate that the dual-phase format is effective in reducinganalyte-analyte interference in a multiplex reaction. Further,consistent with our prior observations, the dual-phase amplificationformat was associated with a significant reduction in the time ofdetection compared with the single-phase format (FIG. 9C).

Results of the T2-ERG amplification in the presence of PCA3 are shown inFIGS. 9D-9F. As shown in FIG. 9D, the single-phase format was able todetect the target template down to ˜500 copies/mL (˜200 copies/r×n),whereas the dual-phase format improved the sensitivity at least 10 foldto ˜50 copies/mL (˜20 copies/r×n) (FIG. 9E). These results alsodemonstrate that the dual-phase format is effective in reducinganalyte-analyte interference in a multiplex reaction. Further,consistent with our prior observations, the dual-phase amplificationformat was associated with a significant reduction in the time ofdetection compared with the single-phase format (FIG. 9F).

Notably, the combined advantages of improved assay sensitivity andprecision, and reduced interference among competing reactions in themultiplex amplification format (i.e., as evidenced by comparison ofsingle analyte and dual analyte performance, such as the performance ofPCA3 alone in a single phase format as shown in FIG. 8A, where 1.1×10³copies yields a strong signal, and the performance of PCA3 in thepresence of T2 in a single phase format as shown in FIG. 9A, where1.25×10³ copies of PCA3 yields a weak signal (due to interference fromT2), and performance of PCA3 in the presence of T2 in a dual phaseformat as shown in FIG. 9B, where 1.25×10³ copies of PCA3 yields a verystrong signal as the result of a significant reduction in theinterference due to T2) was a general feature of the dual-phaseformatted assays. These dramatic advantages would not have beenpredicted in advance of this showing. Additional demonstrations of thisfeature of the dual-phase nucleic acid amplification method follow.

Example 7 Dual-Phase Co-Amplification of PCA3, PSA and T2-ERG

In this example, PCA3, PSA and T2-ERG target templates were co-amplifiedusing the dual-phase forward TMA protocol to determine whether triplexamplification will result in the same improvement in sensitivity andprecision we have observed previously in uniplex and duplex assays(Examples 2-6).

Briefly, T7 primers were hybridized to the target PCA3, PSA and T2-ERGsequences during target capture, followed by removal of excess T7primers. In the first phase of amplification, non-T7 primers were addedalong with all of the requisite amplification, detection and enzymereagents, with the exception of additional T7 primers. After fiveminutes at 42° C., the T7 primers were added to the reaction mixture toinitiate the exponential amplification phase, which was also carried outat 42° C. with real-time detection in three different fluorescentchannels (one for each target). The single-phase control experiment wascarried out using the same primers and detection probes in the standardsingle-phase forward TMA format as described in Example 1.

Results of the PCA3 amplification in the presence of PSA and T2-ERG areshown in FIGS. 10A-10C. As shown in FIG. 10A, the single-phase formatwas able to detect the target template down to ˜12,500 copies/mL (˜5,000copies/r×n), whereas the dual-phase format improved the sensitivity 10fold to ˜1,250 copies/mL (˜500 copies/r×n) (FIG. 10B). Further,consistent with our prior observations, the dual-phase amplificationformat was associated with a significant reduction in the time ofdetection compared with the single-phase format (FIG. 10C).

Results of the T2-ERG amplification in the presence of PCA3 and PSA areshown in FIGS. 10D-10F. As shown in FIG. 10D, the single-phase formatwas able to detect the target template down to ˜5,000 copies/mL (˜2,000copies/r×n), whereas the dual-phase format improved the sensitivity 100fold to ˜50 copies/mL (˜20 copies/r×n) (FIG. 10E). Further, consistentwith our prior observations, the dual-phase amplification format wasassociated with a significant reduction in the time of detectioncompared with the single-phase format (FIG. 10F).

Results of the PSA amplification in the presence of PCA3 and T2-ERG areshown in FIGS. 10G-10I. As shown in FIG. 10G, the single-phase formatwas able to detect the target template down to ˜125,000 copies/mL(˜50,000 copies/r×n), whereas the dual-phase format improved thesensitivity over 10 fold to ˜12,500 copies/mL (˜500 copies/r×n) (FIG.10H). Further, consistent with our prior observations, the dual-phaseamplification format was associated with a significant reduction in thetime of detection compared with the single-phase format (FIG. 10I).

Example 8 Amplification of T2-ERG in Dual-Phase Reverse TMA Format

In this example, we tested a dual-phase reverse TMA protocol usingT2-ERG as a target template. This is in contrast to all of the precedingworking examples, where various forward TMA protocols were employed. Thegeneral description of reverse TMA is set forth above in connection withsingle primer amplification.

In the dual-phase reverse TMA protocol used here, a non-T7 primer washybridized to the 3′ end of the target T2-ERG sequence during targetcapture, followed by removal of excess non-T7 primer. The amplificationprocess was divided into two distinct phases. During the first phase, aT7 promoter provider was introduced along with all of the requisiteamplification, detection and enzyme reagents, with the exception ofadditional non-T7 primer. The T7 promoter provider was blocked at the 3′end, thereby rendering it impossible to extend it enzymatically. In thepresence of reverse transcriptase, the non-T7 primer hybridized to thetarget was extended, creating a cDNA copy, and the target RNA templatewas degraded by the reverse transcriptase's RNase H activity. The T7promoter provider subsequently hybridized to the 3′ end of the cDNA, andthe 3′ end of the cDNA was extended further, filling in the promoterregion of the T7 promoter provider and creating an active,double-stranded template. T7 polymerase then produced multiple RNAtranscripts from the template that were identical to the targettemplate. Because no non-T7 primer was available in the phase 1amplification mixture, the reaction could not proceed any further. Thesecond phase was then started with the addition of non-T7 primer, thusinitiating exponential amplification of the RNA transcript pool producedin phase 1.

Results from the dual-phase reverse TMA experiment are shown in FIG.11B. FIG. 11A shows results of a control single-phase reverse TMAexperiment that was modified to mimic the dual-phase format. Morespecifically, the non-T7 primer was added during the target capture step(allowing the standard 60° C. annealing step to be eliminated from theprotocol); no primers or Enzyme Reagent were added in the first phase;and non-T7 primer and T7 promoter provider as well as Enzyme Reagentwere added to the second phase for the initiation of exponentialamplification. As one can see from FIGS. 11A and 11B, the dual-phasereverse TMA format yielded a significantly improved sensitivity andprecision at the low end of analyte concentration (˜50 copies/r×n)compared with the modified single-phase reverse TMA format. Once again,the dual-phase format yielded superior performance both in terms ofprecision and shorter detection time (FIG. 11C).

Example 9 Co-Amplification of T2-ERG, PCA3, PSA and CAP in Dual-PhaseReverse TMA Format

In this example, T2-ERG, PCA3, PSA and internal control (CAP) targettemplates were co-amplified using two different dual-phase reverse TMAprotocols to determine whether quadruplex amplification will result inthe same improvement in sensitivity and precision we have observedpreviously in uniplex, duplex an triplex assays (Examples 2-8).

Detection of 25 copies/r×n of T2-ERG, which is notoriously difficult toamplify at low levels in the presence of other targets, is shown inFIGS. 12A-12C in the presence of 500,000 copies/r×n of PCA3, 5,000,000copies/r×n of PSA and 5,000 copies/r×n of CAP (open circles) or in thepresence of 5,000 copies/r×n of CAP alone (solid diamonds). FIG. 12Ashows results of a control experiment that was carried out in themodified single-phase reverse TMA format as described above in Example8. FIG. 12B shows results of a dual-phase experiment using thedual-phase reverse TMA format as also described in Example 8. Briefly,non-T7 primers were hybridized to the target T2-ERG, PCA3, PSA and CAPsequences during target capture, followed by removal of excess non-T7primers. In the first phase of amplification, T7 promoter providers wereadded along with all of the requisite amplification, detection andenzyme reagents, with the exception of additional non-T7 primers. Afterfive minutes at 42° C., the non-T7 primers were added to the reactionmixture to initiate the exponential amplification phase, which was alsocarried out at 42° C. with real-time detection in four differentfluorescent channels (one for each target). As one can see from FIGS.12A and 12B, the dual-phase reverse TMA format yielded an improvedsensitivity and precision at 25 copies/r×n T2-ERG in the presence ofPCA3, PSA and CAP and a significantly improved sensitivity and precisionat 25 copies/r×n T2-ERG in the presence of CAP alone compared with themodified single-phase reverse TMA format. These results also demonstratethat the dual-phase format is effective in reducing analyte-analyteinterference in a multiplex reaction. Further, consistent with our priorobservations, the dual-phase amplification format was associated with areduction in the time of detection compared with the single-phaseformat.

FIG. 12C shows results of a different dual-phase format, where in thefirst phase of the reaction PCA3, PSA and CAP (or CAP alone) weresubjected to linear amplification and T2-ERG was subjected toexponential amplification, and in the second phase PCA3, PSA and CAPwere subjected to exponential amplification and T2-ERG continuedamplifying exponentially (all four amplification reaction were carriedout in the same vessel). The distribution of target-specific primersbetween the different phases of amplification is set forth in Table 1below. As one can see from FIGS. 12A and 12C, this different dual-phasereverse TMA format yielded a significantly improved sensitivity at 25copies/r×n T2-ERG in the presence of PCA3, PSA and CAP, or CAP alone,compared with the modified single-phase reverse TMA format. As with thedual-phase format described in previous examples, these resultsdemonstrate that this different dual-phase format is effective inreducing analyte-analyte interference in a multiplex reaction. Further,this different dual-phase amplification format was associated with areduction in the time of detection compared with the single-phaseformat.

TABLE 1 Analyte Target Capture First Phase Second Phase T2-ERG Non-T7primer Non-T7 primer + — T7 promoter provider PCA3 Non-T7 primer T7promoter Non-T7 primer provider PSA Non-T7 primer T7 promoter Non-T7primer provider CAP Non-T7 primer T7 promoter Non-T7 primer provider

Example 10 Co-Amplification of T2-ERG, PCA3, PSA and CAP in Triple-PhaseReverse TMA Format

In this example, T2-ERG, PCA3, PSA and internal control (CAP) targettemplates were co-amplified using dual-phase and triple-phase reverseTMA protocols to determine whether triple-phase amplification mightyield additional improvements in sensitivity and/or precision at the lowend of analyte concentration.

Detection of 150 copies/r×n of T2-ERG is shown in FIGS. 13A-13C in thepresence of 500,000 copies/r×n of PCA3, 5,000,000 copies/r×n of PSA and5,000 copies/r×n of CAP (open circles) or in the presence of 5,000copies/r×n of CAP alone (solid diamonds). FIG. 13A shows results of acontrol experiment that was carried out in the modified single-phasereverse TMA format as described above in Example 8. FIG. 13B showsresults of a dual-phase experiment using the dual-phase reverse TMAformat as also described in Example 8. Sensitivity and precision wereimproved using the dual-phase format.

FIG. 13C shows results of a triple-phase experiment, where in phase 1T2-ERG was subjected to linear amplification and the other 3 analyteswere not amplified, in phase 2 T2-ERG was subjected to exponentialamplification and the 3 other analytes were not amplified, and in phase3 PCA3, PSA and CAP (or CAP alone) were subjected to exponentialamplification and T2-ERG continued amplifying exponentially (all of theamplification reactions proceeded in the same vessel). The distributionof target-specific primers between the different phases of amplificationis set forth in Table 2 below. As one can see from FIGS. 13A and 13C,the triple-phase reverse TMA format yielded vast improvements in bothsensitivity and precision at 150 copies/r×n T2-ERG in the presence ofPCA3, PSA and CAP, or CAP alone, compared with the modified single-phasereverse TMA format. These results also demonstrate that thistriple-phase format is effective in reducing analyte-analyteinterference in a multiplex reaction. Further, this triple-phaseamplification format was associated with a reduction in the time ofdetection compared with the single-phase format.

TABLE 2 Target First Second Third Analyte Capture Phase Phase PhaseT2-ERG Non-T7 T7 promoter Non-T7 — primer provider primer PCA3 Non-T7 —— Non-T7 primer + primer T7 promoter provider PSA Non-T7 — — Non-T7primer + primer T7 promoter provider CAP Non-T7 — — Non-T7 primer +primer T7 promoter provider

As illustrated above, a multiphase amplification format has beendeveloped and demonstrated to work for several different nucleic acidtargets, as well as combinations of targets. Compared to the standardsingle-phase format, the multiphase amplification format resulted insignificant improvements of limits of detection, which will translateinto comparable improvements in the limits of quantitation.

While the present invention has been described and shown in considerabledetail with reference to certain preferred embodiments, those skilled inthe art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe appended claims.

What is claimed is:
 1. A method of quantifying a target nucleic acidsequence in a sample, comprising the steps of: (a) contacting the samplewith a first amplification oligonucleotide, specific for a first portionof the target nucleic acid sequence, under conditions allowinghybridization of the first amplification oligonucleotide to the firstportion of the target nucleic acid sequence, thereby generating apre-amplification hybrid that comprises the first amplificationoligonucleotide and the target nucleic acid sequence; (b) isolating thepre-amplification hybrid by target capture onto a solid support followedby washing to remove any of the first amplification oligonucleotide thatdid not hybridize to the first portion of the target nucleic acidsequence in step (a); (c) amplifying, in a first phase amplificationreaction mixture, at least a portion of the target nucleic acid sequenceof the pre-amplification hybrid isolated in step (b) in a first phase,substantially isothermal, transcription-associated amplificationreaction under conditions that support linear amplification thereof, butdo not support exponential amplification thereof, thereby resulting in areaction mixture comprising a first amplification product, wherein thefirst phase amplification reaction mixture comprises a secondamplification oligonucleotide, the second amplification oligonucleotidebeing complementary to a portion of an extension product of the firstamplification oligonucleotide, and wherein the first amplificationproduct is not a template for nucleic acid synthesis during the firstphase, substantially isothermal, transcription-associated amplificationreaction; (d) combining the reaction mixture comprising the firstamplification product with at least one component that participates inexponential amplification of the first amplification product, but thatis lacking from the reaction mixture comprising the first amplificationproduct, to produce a second phase amplification reaction mixture,wherein the second phase amplification reaction mixture additionallycomprises a sequence-specific hybridization probe; (e) performing, in asecond phase, substantially isothermal, transcription-associatedamplification reaction in the second phase amplification reactionmixture, an exponential amplification of the first amplificationproduct, thereby synthesizing a second amplification product; (f)detecting, with the sequence-specific hybridization probe at regulartime intervals, synthesis of the second amplification product in thesecond phase amplification reaction mixture; and (g) quantifying thetarget nucleic acid sequence in the sample using results from step (f).2. The method of claim 1, wherein the first amplificationoligonucleotide comprises a 3′ target specific sequence and a 5′promoter sequence for an RNA polymerase.
 3. The method of claim 2,wherein the RNA polymerase is T7 RNA polymerase.
 4. The method of claim1, wherein the second amplification oligonucleotide is enzymaticallyextended in the first phase isothermal transcription-associatedamplification reaction.
 5. The method of claim 1, wherein the solidsupport comprises an immobilized capture probe.
 6. The method of claim1, wherein step (a) further comprises contacting the sample with atarget capture oligonucleotide that hybridizes to the target nucleicacid sequence, and wherein the pre-amplification hybrid comprises thetarget nucleic acid sequence hybridized to each of the target captureoligonucleotide and the first amplification oligonucleotide.
 7. Themethod of claim 1, wherein the solid support comprises magneticallyattractable particles.
 8. The method of claim 1, wherein each of thefirst and second phase isothermal transcription-associated amplificationreactions comprise an RNA polymerase and a reverse transcriptase, andwherein the reverse transcriptase comprises an endogenous RNaseHactivity.
 9. The method of claim 1, wherein the at least one componentcomprises the first amplification oligonucleotide.
 10. The method ofclaim 1, wherein the first amplification product of step (c) is a cDNAmolecule with the same polarity as the target nucleic acid sequence inthe sample, and wherein the second amplification product of step (e) isan RNA molecule.
 11. The method of claim 1, wherein thesequence-specific hybridization probe in step (d) is aconformation-sensitive probe that produces a detectable signal whenhybridized to the second amplification product.
 12. The method of claim1, wherein the sequence-specific hybridization probe in step (d) is afluorescently labeled sequence-specific hybridization probe.
 13. Themethod of claim 1, wherein step (g) comprises quantifying the targetnucleic acid sequence in the sample using a linear calibration curve andresults from step (f).
 14. The method of claim 1, wherein step (c)comprises amplifying by 10-fold to 10,000-fold, in the first phaseamplification reaction mixture.